Patent application title: Methods Of Intracellular Conversion Of Single-Chain Proteins Into Their Di-Chain Form
Inventors:
Sanjiv Ghanshani (Irvine, CA, US)
Sanjiv Ghanshani (Irvine, CA, US)
Linh Q. Le (Tustin, CA, US)
Yi Liu (Irvine, CA, US)
Lance E. Steward (Irvine, CA, US)
Assignees:
Allergan, Inc.
IPC8 Class: AC12P2100FI
USPC Class:
435 681
Class name: Chemistry: molecular biology and microbiology micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition enzymatic production of a protein or polypeptide (e.g., enzymatic hydrolysis, etc.)
Publication date: 2014-01-30
Patent application number: 20140030759
Abstract:
The present specification discloses expression constructs comprising
single-chain proteins comprising a di-chain loop region comprising an
exogenous protease cleavage site and a protease that can cleave the
exogenous protease cleavage site located within the di-chain loop, cell
compositions comprising such expression construct, and intracellular
methods of converting the single-chain protein into its di-chain form.Claims:
1. An intracellular method of converting a single-chain protein into its
di-chain form, the method comprising the steps of: a) growing a cell
comprising a dual expression construct at a first temperature for a
certain period of time in order to achieve maximal cell density, the dual
expression construct comprising; i) an open reading frame encoding a
single-chain protein comprising a di-chain loop region comprising an
exogenous protease cleavage site; and ii) an open reading frame encoding
a protease; wherein the protease can cleave the exogenous protease
cleavage site located within the di-chain loop; b) growing the cell at a
second temperature for a certain period of time in order to achieve
maximal induction of protein expression from the open reading frame
encoding the single-chain protein, wherein growth at step (b) induces
expression of the single-chain protein and the protease from the dual
expression construct; and wherein the produced protease cleaves the
single-chain protein at the exogenous protease cleavage site located
within the di-chain loop region, thereby converting the single-chain
protein into its di-chain form.
2. An intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of: a) growing a cell comprising a dual expression construct at 37.degree. C. for about 3.5 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; and ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at 22.degree. C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain Clostridial toxin and the protease from the dual expression construct; and wherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.
3. The method according to 2, wherein the exogenous protease cleavage site is an enterokinase protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a Tobacco Vein Mottling Virus (TVMV) protease cleavage site, a subtilisin protease cleavage site, or a Caspase 3 protease cleavage site.
Description:
[0001] This application is a continuation and claims priority pursuant to
35 U.S.C. §120 to U.S. patent application Ser. No. 13/575,222, filed
Jul. 25, 2012, which is a national stage application under 35 U.S.C.
§371 of PCT patent application PCT/US2011/022272, filed on Jan. 24,
2011, which claims the benefit of U.S. Provisional Patent Application
61/286,963 filed Jan. 25, 2010, each of which is hereby incorporated by
reference in its entirety.
[0002] The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to inhibit neuronal transmission are being exploited in a wide variety of therapeutic and cosmetic applications, see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc., 2004). Clostridial toxins commercially available as pharmaceutical compositions include, BoNT/A preparations, such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.), DYSPORT®/RELOXIN®, (Beaufour Ipsen, Porton Down, England), NEURONOX® (Medy-Tox, Inc., Ochang-myeon, South Korea) BTX-A (Lanzhou Institute Biological Products, China) and XEOMIN® (Merz Pharmaceuticals, GmbH., Frankfurt, Germany); and BoNT/B preparations, such as, e.g., MYOBLOC®/NEUROBLOC® (Elan Pharmaceuticals, San Francisco, Calif.). As an example, BOTOX® is currently approved in one or more countries for the following indications: achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and VII nerve disorder.
[0003] The therapeutic utility of Clostrdial toxins has been expanded beyond their current myo-relaxant applications to treat sensory nerve-based ailments, such as, e.g., various kinds of chronic pain, neurogenic inflammation and urogentital disorders, as well as non-neuronal-based disorders, such as, e.g., pancreatitis. One approach that is currently being exploited to expand Clostridial toxin-based therapies involves modifying a Clostridial toxin so that the modified toxin has an altered cell targeting capability for a non-Clostridial toxin target cell. This re-targeted capability is achieved by replacing a naturally-occurring targeting domain of a Clostridial toxin with a targeting domain showing a selective binding activity for a non-Clostridial toxin receptor present in a non-Clostridial toxin target cell. Such modifications to a targeting domain result in a modified toxin that is able to selectively bind to a non-Clostridial toxin receptor (target receptor) present on a non-Clostridial toxin target cell (re-targeted). A re-targeted Clostridial toxin with a targeting activity for a non-Clostridial toxin target cell can bind to a receptor present on the non-Clostridial toxin target cell, translocate into the cytoplasm, and exert its proteolytic effect on the SNARE complex of the non-Clostridial toxin target cell.
[0004] Non-limiting examples of re-targeted Clostridial toxins with a targeting activity for a non-Clostridial toxin target cell are described in, e.g., Keith A. Foster et al., Clostridial Toxin Derivatives Able To Modify Peripheral Sensory Afferent Functions, U.S. Pat. No. 5,989,545; Clifford C. Shone et al., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617; Stephan Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 6,500,436; Conrad P. Quinn et al., Methods and Compounds for the Treatment of Mucus Hypersecretion, U.S. Pat. No. 6,632,440; Lance E. Steward et al., Methods And Compositions For The Treatment Of Pancreatitis, U.S. Pat. No. 6,843,998; J. Oliver Dolly et al., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,419,676; Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. Pat. No. 7,514,088; Keith A. Foster et al., Inhibition of Secretion from Non-neural Cells, U.S. Patent Publication 2003/0180289; and Keith A. Foster et al., Re-targeted Toxin Conjugates, International Patent Publication WO 2005/023309. The ability to re-target the therapeutic effects associated with Clostridial toxins has greatly extended the number of medicinal applications able to use a Clostridial toxin therapy. As a non-limiting example, modified Clostridial toxins retargeted to sensory neurons are useful in treating various kinds of chronic pain, such as, e.g., hyperalgesia and allodynia, neuropathic pain and inflammatory pain, see, e.g., Foster, supra, (1999); and Donovan, supra, (2002); and Stephan Donovan, Method For Treating Neurogenic Inflammation Pain with Botulinum Toxin and Substance P Components, U.S. Pat. No. 7,022,329. As another non-limiting example, modified Clostridial toxins retargeted to pancreatic cells are useful in treating pancreatitis, see, e.g., Steward, supra, (2005).
[0005] Clostridial toxins, whether naturally occurring or modified, are processed into a di-chain form in order to achieve full activity. Naturally-occurring Clostridial toxins are each translated as a single-chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease (FIG. 1). This cleavage occurs within the discrete di-chain loop region created between two cysteine residues that form a disulfide bridge. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC), comprising the enzymatic domain, and an approximately 100 kDa heavy chain (HC), comprising the translocation and cell binding domains, the LC and HC being held together by the single disulfide bond and non-covalent interactions (FIG. 1). Recombinantly-produced Clostridial toxins generally substitute the naturally-occurring di-chain loop protease cleavage site with an exogenous protease cleavage site (FIG. 2). See e.g., Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat. No. 7,419,676, which is hereby incorporated by reference. Although re-targeted Clostridial toxins vary in their overall molecular weight because of the size of the targeting moiety, the activation process and its reliance on exogenous cleavage sites is essentially the same as that for recombinantly-produced Clostridial toxins. See e.g., Steward, L. E. et al., Activatable Clostridial Toxins, U.S. Patent Publication 2009/0005313; Steward, L. E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S. patent application Ser. No. 11/776,075; Steward, L. E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity for Clostridial Toxin Target Cells, U.S. Patent Publication 2008/0241881, each of which is hereby incorporated by reference.
[0006] To date, the conversion of the single-chain form of a recombinantly produced Clostridial toxin or modified Clostridial toxin into its di-chain form required an in vitro activation process. First, the bacterial cells used to recombinantly produce these toxins lack the naturally-occurring protease present in the Clostridial strains that produce the native toxins. Second, there has been no great need for bacterial cells to produce activated toxins recombinantly because of safety concerns raised in handling activated toxins. See e.g., Dolly, U.S. Pat. No. 7,419,676, supra, (2008). However, if these concerns could be overcome, the production of recombinantly produced activated toxins would streamline the manufacturing process of recombinantly produced Clostridial toxins or modified Clostridial toxins. For example, currently the manufacture of recombinantly produced Clostridial toxins or modified Clostridial toxins involves the following purification steps: 1) immobilized metal affinity chromatography, 2) buffer exchange dialysis, 3) protease cleavage reaction, 4), ion exchange chromatography and 5) addition of PEG and flash freezing for storage at -80° C. The use of a bacterial cell that can protealytically cleave the recombinant Clostridial toxin intracellularly while it is still expressing the toxin can reduce the number of purification steps to the following: 1) immobilized metal affinity chromatography, 2) buffer exchange dialysis, 3) ion exchange chromatography, and 4) addition of PEG and flash freezing for storage at -80° C.
[0007] The present specification discloses a method of converting a single-chain protein comprising a di-chain loop region into its di-chain form that does not rely on an in vitro process for converting the single-chain form of the toxin into its di-chain form. This is accomplished by the use of cells that express both the protein and the protease necessary to convert it to active di-chain.
[0008] Thus, aspects of the present specification provide, a dual expression construct that includes an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. Polynucleotides, as well as the Clostridial toxins comprising a di-chain loop region comprising an exogenous protease cleavage site that they encode, are described in, e.g., Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat. No. 7,132,259; Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat. No. 7,419,676, each of which is hereby incorporated by reference in its entirety. Polynucleotides, as well as the proteins comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site that they encode, are described in, e.g., Steward, L. E. et al., Multivalent Clostridial Toxins, U.S. Patent Publication 2009/0048431; Steward, L. E. et al., Activatable Clostridial Toxins, U.S. Patent Publication 2009/0069238; Steward, L. E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S. patent application Ser. No. 11/776,075; Steward, L. E. et al., Modified Clostridial Toxins with Enhanced Translocation Capabilities and Altered Targeting Activity for Clostridial Toxin Target Cells, U.S. Patent Publication 2008/0241881; Foster, K. A. et al., Fusion Proteins, U.S. Patent Publication 2009/0035822; Foster, K. A. et al., Non-Cytotoxic Protein Conjugates, U.S. Patent Publication 2009/0162341; Steward, L. E. et al., Activatable Clostridial Toxins, U.S. Patent Publication 2008/0032931; Foster, K. A. et al., Non-Cytotoxic Protein Conjugates, U.S. Patent Publication 2008/0187960; Steward, L. E. et al., Degradable Clostridial Toxins, U.S. Patent Publication 2008/0213830; Steward, L. E. et al., Modified Clostridial Toxins With Enhanced Translocation Capabilities and Altered Targeting Activity For Clostridial Toxin Target Cells, U.S. Patent Publication 2008/0241881; Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat. No. 7,419,676; and a companion patent application Ghanshani, et al., Modified Clostridial Toxins Comprising an Integrated Protease Cleavage Site-Binding Domain, Attorney Docket No. 18468 PROV (BOT), each of which is hereby incorporated by reference in its entirety.
[0009] Other aspects of the present specification provide a cell comprising a dual expression construct that includes an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
[0010] Yet other aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at a first temperature for a certain period of time in order to achieve maximal cell density, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; and ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein, wherein growth at step (b) induces expression of the single-chain protein and the protease from the dual expression construct; and wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0011] Still other aspects of the present specification provide an intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of: a) growing a cell comprising a dual expression construct at 37° C. for about 2 to about 3.5 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; and ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at 22° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain Clostridial toxin and the protease from the dual expression construct; and wherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.
[0012] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37° C. for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 12 to about 16° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0013] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37° C. for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 20 to about 24° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0014] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37° C. for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 12 to about 16° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0015] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising a dual expression construct at 37° C. for about 2 to about 8 hours, the dual expression construct comprising; i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site; and ii) an open reading frame encoding a TEV protease; b) growing the cell at about 20 to about 24° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0016] Other aspects of the present specification provide, an expression construct comprising an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
[0017] Other aspects of the present specification provide, an expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site.
[0018] Other aspects of the present specification provide a cell comprising an expression construct comprising an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and another expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site. In further aspects, the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site can be, e.g., a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, a modified Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site, or a single-chain protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
[0019] Yet other aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing a cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site and ii) another expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; b) growing the cell at a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein, wherein growth at step (b) induces expression of the single-chain protein and the protease from the expression constructs; and wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0020] Still other aspects of the present specification provide an intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of: a) growing at 37° C. for about 2 to about 3.5 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site and ii) another expression construct comprising an open reading frame encoding a protease that can proteolytically cleave the exogenous protease cleavage site located in the di-chain loop region of a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; b) growing the cell at 22° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain Clostridial toxin and the protease from the expression constructs; and wherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.
[0021] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37° C. for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 12 to about 16° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0022] Further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37° C. for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, and an integrated TEV protease cleavage site-opioid binding domain and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 20 to about 24° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0023] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37° C. for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 12 to about 16° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0024] Yet further aspects of the present specification provide an intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of a) growing at 37° C. for about 2 to about 8 hours a cell, the cell comprising i) an expression construct comprising an open reading frame encoding a single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site and ii) another expression construct comprising an open reading frame encoding TEV protease; b) growing the cell at about 20 to about 24° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the expression constructs; and wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the domain organization of naturally-occurring Clostridial toxins. The single chain form depicts the amino to carboxyl linear organization comprising an enzymatic domain, a translocation domain, and a HC binding domain. The di-chain loop region located between the translocation and enzymatic domains is depicted by the double SS bracket. This region comprises an endogenous di-chain loop protease cleavage site that upon proteolytic cleavage with a naturally-occurring protease, such as, e.g., an endogenous Clostridial toxin protease or a naturally-occurring protease produced in the environment, converts the single chain form of the toxin into the di-chain form.
[0026] FIG. 2 shows a schematic of the current paradigm of neurotransmitter release and Clostridial toxin intoxication in a central and peripheral neuron. FIG. 2a shows a schematic for the neurotransmitter release mechanism of a central and peripheral neuron. The release process can be described as comprising two steps: 1) vesicle docking, where the vesicle-bound SNARE protein of a vesicle containing neurotransmitter molecules associates with the membrane-bound SNARE proteins located at the plasma membrane; and 2) neurotransmitter release, where the vesicle fuses with the plasma membrane and the neurotransmitter molecules are exocytosed. FIG. 2b shows a schematic of the intoxication mechanism for tetanus and botulinum toxin activity in a central and peripheral neuron. This intoxication process can be described as comprising four steps: 1) receptor binding, where a Clostridial toxin binds to a Clostridial receptor system and initiates the intoxication process; 2) complex internalization, where after toxin binding, a vesicle containing the toxin/receptor system complex is endocytosed into the cell; 3) light chain translocation, where multiple events are thought to occur, including, e.g., changes in the internal pH of the vesicle, formation of a channel pore comprising the HN domain of the Clostridial toxin heavy chain, separation of the Clostridial toxin light chain from the heavy chain, and release of the active light chain and 4) enzymatic target modification, where the activate light chain of Clostridial toxin proteolytically cleaves its target SNARE substrate, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking and neurotransmitter release.
[0027] Clostridia toxins produced by Clostridium botulinum, Clostridium tetani, Clostridium baratii and Clostridium butyricum are the most widely used in therapeutic and cosmetic treatments of humans and other mammals. Strains of C. botulinum produce seven antigenically-distinct types of Botulinum toxins (BoNTs), which have been identified by investigating botulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and /D), or isolated from soil (BoNT/G). BoNTs possess approximately 35% amino acid identity with each other and share the same functional domain organization and overall structural architecture. It is recognized by those of skill in the art that within each type of Clostridial toxin there can be subtypes that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4, with specific subtypes showing approximately 89% amino acid identity when compared to another BoNT/A subtype. While all seven BoNT serotypes have similar structure and pharmacological properties, each also displays heterogeneous bacteriological characteristics. In contrast, tetanus toxin (TeNT) is produced by a uniform group of C. tetani. Two other Clostridia species, C. baratii and C. butyricum, produce toxins, BaNT and BuNT, which are similar to BoNT/F and BoNT/E, respectively.
[0028] Each mature di-chain molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets core components of the neurotransmitter release apparatus; 2) a translocation domain (HN) contained within the amino-terminal half of the HC that facilitates release of the LC from intracellular vesicles into the cytoplasm of the target cell; and 3) a binding domain (HC) found within the carboxyl-terminal half of the HC that determines the binding activity and binding specificity of the toxin to the receptor complex located at the surface of the target cell. The HC domain comprises two distinct structural features of roughly equal size that indicate function and are designated the HCN and HCC subdomains. Table 1 gives approximate boundary regions for each domain found in exemplary Clostridial toxins.
TABLE-US-00001 TABLE 1 Clostridial Toxin Reference Sequences and Regions Toxin SEQ ID NO: LC HN HC BoNT/A 1 M1-K448 A449-K871 N872-L1296 BoNT/B 2 M1-K441 A442-S858 E859-E1291 BoNT/C1 3 M1-K449 T450-N866 N867-E1291 BoNT/D 4 M1-R445 D446-N862 S863-E1276 BoNT/E 5 M1-R422 K423-K845 R846-K1252 BoNT/F 6 M1-K439 A440-K864 K865-E1274 BoNT/G 7 M1-K446 S447-S863 N864-E1297 TeNT 8 M1-A457 S458-V879 I880-D1315 BaNT 9 M1-K431 N432-I857 I858-E1268 BuNT 10 M1-R422 K423-I847 K848-K1251
[0029] The binding, translocation, and enzymatic activity of these three functional domains are all necessary for toxicity. While all details of this process are not yet precisely known, the overall cellular intoxication mechanism whereby Clostridial toxins enter a neuron and inhibit neurotransmitter release is similar, regardless of serotype or subtype. Although the applicants have no wish to be limited by the following description, the intoxication mechanism can be described as comprising at least four steps: 1) receptor binding, 2) complex internalization, 3) light chain translocation, and 4) enzymatic target modification (FIG. 3). The process is initiated when the HC domain of a Clostridial toxin binds to a toxin-specific receptor system located on the plasma membrane surface of a target cell. The binding specificity of a receptor complex is thought to be achieved, in part, by specific combinations of gangliosides and protein receptors that appear to distinctly comprise each Clostridial toxin receptor complex. Once bound, the toxin/receptor complexes are internalized by endocytosis and the internalized vesicles are sorted to specific intracellular routes. The translocation step appears to be triggered by the acidification of the vesicle compartment. This process seems to initiate two important pH-dependent structural rearrangements that increase hydrophobicity and promote formation of the di-chain form of the toxin. Once activated, light chain endopeptidase of the toxin is released from the intracellular vesicle into the cytosol where it appears to specifically target one of three known core components of the neurotransmitter release apparatus. These core proteins, vesicle-associated membrane protein (VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking and fusion at the nerve terminal and constitute members of the soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminal region, releasing a nine or twenty-six amino acid segment, respectively, and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conserved central portion of VAMP, and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1 cleaves syntaxin at a single site near the cytosolic membrane surface. The selective proteolysis of synaptic SNAREs accounts for the block of neurotransmitter release caused by Clostridial toxins in vivo. The SNARE protein targets of Clostridial toxins are common to exocytosis in a variety of non-neuronal types; in these cells, as in neurons, light chain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al., Botulinum and Tetanus Neurotoxins: Structure, Function and Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437, (2003).
[0030] In an aspect of the invention, a modified Clostridial toxin comprises, in part, a single-chain modified Clostridial toxin and a di-chain modified Clostridial toxin. As discussed above, a Clostridial toxin, whether naturally-occurring or non-naturally-occurring, are initially synthesized as a single-chain polypeptide. This single-chain form is subsequently cleaved at a protease cleavage site located within a discrete di-chain loop region created between two cysteine residues that form a disulfide bridge by a protease. This posttranslational processing yields a di-chain molecule comprising a light chain (LC) and a heavy chain. As used herein, the term "di-chain loop region" refers to loop region of a naturally-occurring or non-naturally-occurring Clostridial toxin formed by a disulfide bridge located between the LC domain and the HC domain. As used herein, the term "single-chain modified Clostridial toxin" refers to any modified Clostridial toxin disclosed in the present specification that is in its single-chain form, i.e., the toxin has not been cleaved at the protease cleavage site located within the di-chain loop region by its cognate protease. As used herein, the term "di-chain modified Clostridial toxin" refers to any modified Clostridial toxin disclosed in the present specification that is in its di-chain form, i.e., the toxin has been cleaved at the protease cleavage site located within the di-chain loop region by its cognate protease.
[0031] Aspects of the present invention provide, in part, polynucleotide molecules. As used herein, the term "polynucleotide molecule" is synonymous with "nucleic acid molecule" and means a polymeric form of nucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, of any length. Useful polynucleotide molecules, include, without limitation, naturally-occurring and non-naturally-occurring DNA molecules and naturally-occurring and non-naturally-occurring RNA molecules. Non-limiting examples of naturally-occurring and non-naturally-occurring DNA molecules include single-stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules, cDNA molecules, vector constructs, such as, e.g., plasmid constructs, phagemid constructs, bacteriophage constructs, retroviral constructs and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non-naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA and mRNA.
[0032] Well-established molecular biology techniques that may be necessary to make a polynucleotide molecule encoding a modified Clostridial toxin disclosed in the present specification include, but not limited to, procedures involving polymerase chain reaction (PCR) amplification, restriction enzyme reactions, agarose gel electrophoresis, nucleic acid ligation, bacterial transformation, nucleic acid purification, nucleic acid sequencing and recombination-based techniques are routine and well within the scope of one skilled in the art and from the teaching herein. Non-limiting examples of specific protocols necessary to make a polynucleotide molecule encoding a modified Clostridial toxin are described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004). Additionally, a variety of commercially available products useful for making a polynucleotide molecule encoding a modified Clostridial toxin are widely available. These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.
[0033] The methods disclosed in the present specification include, in part, an open reading frame. As used herein, the term "open reading frame" is synonymous with "ORF" and means any polynucleotide molecule that encodes a protein, or a portion of a protein. An open reading frame usually begins with a start codon (represented as, e.g. AUG for an RNA molecule and ATG in a DNA molecule in the standard code) and is read in codon-triplets until the frame ends with a STOP codon (represented as, e.g. UAA, UGA or UAG for an RNA molecule and TAA, TGA or TAG in a DNA molecule in the standard code). As used herein, the term "codon" means a sequence of three nucleotides in a polynucleotide molecule that specifies a particular amino acid during protein synthesis; also called a triplet or codon-triplet.
[0034] The methods disclosed in the present specification include, in part, an expression construct. An expression construct comprises a polynucleotide molecule including an open reading frame disclosed in the present specification operably-linked to an expression vector useful for expressing the polynucleotide molecule in a cell or cell-free extract. A wide variety of expression vectors can be employed for expressing a polynucleotide molecule disclosed in the present specification, including, without limitation, a viral expression vector; a prokaryotic expression vector; eukaryotic expression vectors, such as, e.g., a yeast expression vector, an insect expression vector and a mammalian expression vector; and a cell-free (in vitro) expression vector. It is further understood that expression vectors useful to practice aspects of these methods may include those which express the polynucleotide molecule under control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.
[0035] The expression constructs disclosed in the present specification can comprise an open reading frame encoding a protein including a di-chain loop region comprising an exogenous protease cleavage site, wherein cleavage of the exogenous protease cleavage site converts the single-chain protein into its di-chain form. In aspects of this embodiment, a viral expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; a prokaryotic expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; a yeast expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; an insect expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop; and a mammalian expression vector is operably-linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop. In other aspects of this embodiment, an expression construct--suitable for expressing a polynucleotide molecule disclosed in the present specification can be expressed using a cell-free extract. In an aspect of this embodiment, a cell-free expression vector is operably linked to a polynucleotide molecule encoding a protein comprising an exogenous protease cleavage site located within the di-chain loop.
[0036] In an embodiment, an expression construct disclosed in the present specification can comprise an open reading frame encoding a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site. In aspects of this embodiment, an expression construct disclosed in the present specification can comprise an open reading frame encoding a Clostridial toxin comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In aspects of this embodiment, the single-chain Clostridial toxin comprises a linear amino-to-carboxyl order of 1) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial translocation domain and the Clostridial binding domain; 2) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial binding domain and the Clostridial translocation domain; 3) the Clostridial binding domain, the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin enzymatic domain; 4) the Clostridial binding domain, the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin translocation domain; 5) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin enzymatic domain and the Clostridial binding domain; or 6) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial binding domain and the Clostridial toxin enzymatic domain.
[0037] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a BoNT/A toxin enzymatic domain, a BoNT/A translocation domain, a BoNT/A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a BoNT/B enzymatic domain, a BoNT/B translocation domain, a BoNT/B binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain, a BoNT/C1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a BoNT/D enzymatic domain, a BoNT/D translocation domain, a BoNT/D binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a BoNT/E enzymatic domain, a BoNT/E translocation domain, a BoNT/E binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a BoNT/F enzymatic domain, a BoNT/F translocation domain, a BoNT/F binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a BoNT/G enzymatic domain, a BoNT/G translocation domain, a BoNT/G binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 8) a TeNT enzymatic domain, a TeNT translocation domain, a TeNT binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 9) a BaNT enzymatic domain, a BaNT translocation domain, a BaNT binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 10) a BuNT enzymatic domain, a BuNT translocation domain, a BuNT binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0038] In further other aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a BoNT/A toxin enzymatic domain, a BoNT/A translocation domain, a BoNT/A binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 2) a BoNT/B enzymatic domain, a BoNT/B translocation domain, a BoNT/B binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 3) a BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain, a BoNT/C1 binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 4) a BoNT/D enzymatic domain, a BoNT/D translocation domain, a BoNT/D binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 5) a BoNT/E enzymatic domain, a BoNT/E translocation domain, a BoNT/E binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 6) a BoNT/F enzymatic domain, a BoNT/F translocation domain, a BoNT/F binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 7) a BoNT/G enzymatic domain, a BoNT/G translocation domain, a BoNT/G binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 8) a TeNT enzymatic domain, a TeNT translocation domain, a TeNT binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 9) a BaNT enzymatic domain, a BaNT translocation domain, a BaNT binding domain, and a di-chain loop region comprising a TEV protease cleavage site; 10) a BuNT enzymatic domain, a BuNT translocation domain, a BuNT binding domain, and a di-chain loop region comprising a TEV protease cleavage site.
[0039] Examples of such Clostridial toxins comprising a di-chain loop region comprising an exogenous protease cleavage sit are described in, e.g., J. Oliver Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,132,529; J. Oliver Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,419,676; Lance Steward, et al., Leucine-Based Motifs and Clostridial Neurotoxins, U.S. Pat. No. 6,903,187; Lance Steward, et al., Leucine-Based Motifs and Clostridial Neurotoxins, U.S. Pat. No. 7,393,925; Wei-Jen Lin, et al., Neurotoxins with Enhanced Target Specificity, U.S. Pat. No. 7,273,722; Lance Steward, et al., Modified Botulinum Neurotoxins, U.S. Pat. No. 7,491,799; Lance E. Steward, et al., Optimized Expression of Active Botulinum Toxin Type E, U.S. Patent Publication 2008/0138893; Ester Fernandez-Salas, et al., Optimized Expression of Active Botulinum Toxin Type A, U.S. Patent Publication 2008/0057575; each of which is hereby incorporated by reference in its entirety.
[0040] In another embodiment, an expression construct disclosed in the present specification can comprise an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In aspects of this embodiment, the single-chain protein comprises a linear amino-to-carboxyl order of 1) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial translocation domain and the non-Clostridial binding domain; 2) the Clostridial enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the non-Clostridial binding domain and the Clostridial translocation domain; 3) the non-Clostridial binding domain, the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin enzymatic domain; 4) the non-Clostridial binding domain, the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin translocation domain; 5) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin enzymatic domain and the non-Clostridial binding domain; or 6) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the non-Clostridial binding domain and the Clostridial toxin enzymatic domain.
[0041] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an opioid binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an enkephalin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a bovine adrenomedullary-22 (BAM22) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an endomorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an endorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a dynorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a nociceptin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a hemorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a rimorphin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0042] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a melanocortin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an melanocyte stimulating hormone binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an adrenocorticotropin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a lipotropin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0043] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a galanin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a galanin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a galanin message-associated peptide (GMAP) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0044] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a granin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a chromogranin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a chromogranin B binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a chromogranin C binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0045] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a tachykinin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Substance P binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuropeptide K binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuropeptide gamma binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurokinin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a hemokinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a endokinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0046] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Neuropeptide Y related peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuropeptide Y (NPY) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Peptide YY (PYY) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Pancreatic peptide (PP) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Pancreatic icosapeptide (PIP) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0047] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurohormone peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a corticotropin-releasing hormone (CCRH) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a parathyroid hormone (PTH) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a thyrotropin-releasing hormone (TRH) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a somatostatin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0048] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cytokine peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a ciliary neurotrophic factor (CNTF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a glycophorin-A (GPA) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a leukemia inhibitory factor (LIF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an interleukin (IL) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an onostatin M binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cardiotrophin-1 (CT-1) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cardiotrophin-like cytokine (CLC) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neuroleukin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0049] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a kinin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a bradykinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a kallidin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a desArg9 bradykinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a desArg10 bradykinin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0050] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Fibroblast growth factor (FGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-4 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-8 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-9 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-17 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a FGF-18 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0051] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurotrophin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a nerve growth factor (NGF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a brain derived neurotrophic factor (BDNF) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurotrophin-3 (NT-3) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurotrophin-4/5 (NT-4/5) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a head activator peptide (HA) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0052] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a tumor necrosis factor (TNF)peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0053] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Glial derived growth factor (GDNF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a neurturin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a persephrin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an artemin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0054] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Transformation growth factor β (TGFβ) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFβ1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFβ2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFβ3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a TGFβ4 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0055] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Bone morphogenetic protein β (BMP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP4 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP5 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP6 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP7 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP8 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a BMP10 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0056] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Growth differentiation factor β (GDF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF5 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF6 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF7 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF8 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF10 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 9) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF11 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 10) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a GDF15 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0057] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin B binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin C binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an activin E binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an inhibin A binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0058] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Vascular endothelial growth factor (VEGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0059] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an insulin growth factor (IGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an IGF-1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an IGF-2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0060] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an Epidermal growth factor (EGF) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0061] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Glucagon like hormone peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a secretin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a glucagon-like peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0062] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Pituitary adenylate cyclase activating peptide (PACAP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0063] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Growth hormone-releasing hormone (GHRH) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0064] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Growth hormone-releasing hormone (GHRH) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0065] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Vasoactive intestinal peptide (VIP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a VIP1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a VIP2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0066] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Gastric inhibitory polypeptide (GIP) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0067] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Calcitonin-related peptidesvisceral gut peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a gastrin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a gastrin-releasing peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a cholecystokinin (CCK) binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0068] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a protease activated receptor (PAR) peptide binding domain, and a di-chain loop region comprising an exogenous protease cleavage site. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR1 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR2 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; or 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a PAR3 binding domain, and a di-chain loop region comprising an exogenous protease cleavage site.
[0069] Examples, of such proteins comprising a di-chain loop region comprising an exogenous protease cleavage site are described in, e.g., J. Oliver Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,132,529; J. Oliver Dolly, et al., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,419,676; Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. Pat. No. 7,514,088; Keith A. Foster et al., Re-targeted Toxin Conjugates, International Patent Publication WO 2005/023309; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2008/0032930; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2008/0032931; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2008/0161226; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2008/0221012; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0004224; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0005313; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0018081; Lance E. Steward, et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication 2009/0069238; and Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives and Methods of Their Use, U.S. Patent Publication 2009/0048431, each of which is hereby incorporated by reference in its entirety.
[0070] In another embodiment, an expression construct comprises an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated protease cleavage site-binding domain. In aspects of this embodiment, the single-chain protein comprises a linear amino-to-carboxyl order of 1) an integrated protease cleavage site-binding domain, a Clostridial toxin translocation domain and a Clostridial toxin enzymatic domain; 2) an integrated protease cleavage site-binding domain, a Clostridial toxin enzymatic domain, and a Clostridial toxin translocation domain; 3) a Clostridial toxin enzymatic domain, an integrated protease cleavage site-binding domain, and a Clostridial toxin translocation domain; 4) a Clostridial toxin translocation domain, an integrated protease cleavage site-binding domain, and a Clostridial toxin enzymatic domain; 5) a Clostridial toxin translocation domain, a Clostridial toxin enzymatic domain, and an integrated protease cleavage site-binding domain; and 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated protease cleavage site-binding domain.
[0071] In other aspects of this embodiment, an expression construct comprises an open reading frame encoding a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-opioid binding domain. In further aspects of this embodiment, an expression construct comprises an open reading frame encoding 1) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-enkephalin binding domain; 2) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-bovine adrenomedullary-22 (BAM22) binding domain; 3) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-endomorphin binding domain; 4) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-endorphin binding domain; 5) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-dynorphin binding domain; 6) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-nociceptin binding domain; 7) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-hemorphin binding domain; or 8) a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-rimorphin binding domain
[0072] Examples, of such proteins comprising integrated protease cleavage site-binding domain are described in, e.g., companion patent application Sanjiv Ghanshani, et al., Modified Clostridial Toxins Comprising an Integrated Protease Cleavage Site-Binding Domain, which is hereby incorporated by reference in its entirety.
[0073] The expression constructs disclosed in the present specification can comprise an open reading frame encoding a protease. In aspects of this embodiment, a viral expression vector is operably-linked to a polynucleotide molecule encoding a protease; a prokaryotic expression vector is operably-linked to a polynucleotide molecule encoding a protease; a yeast expression vector is operably-linked to a polynucleotide molecule encoding a protease; an insect expression vector is operably-linked to a polynucleotide molecule encoding a protease; and a mammalian expression vector is operably-linked to a polynucleotide molecule encoding a protease. In other aspects of this embodiment, an expression construct is suitable for expressing a polynucleotide molecule disclosed in the present specification can be expressed using a cell-free extract. In an aspect of this embodiment, a cell-free extract expression vector is operably linked to a polynucleotide molecule encoding a protease.
[0074] In aspect of this embodiment, an expression construct comprising an open reading frame encodes an enterokinase, a human rhinovirus 3C protease, a human enterovirus 3C protease, a tobacco etch virus (TEV) protease, a Tobacco Vein Mottling Virus (TVMV) protease, a subtilisin protease, or a Caspase 3 protease. Examples of Enterokinase proteases and the polynucleotide molecules that encode them are described in, e.g., Edward R. LaVallie, Cloning of Enterokinase and Method of Use, U.S. Pat. No. 5,665,566; Edward R. LaVallie, Cloning of Enterokinase and Method of Use, U.S. Pat. No. 6,746,859, each of which is hereby incorporated by reference in its entirety. Examples of subtilisin proteases and the polynucleotide molecules that encode them are described in, e.g., Donn N. Rubingh, et al., Subtillisin Protease Variants having Amino Acid Deletions and Substitutions in Defined Epitope Regions, U.S. Pat. No. 6,586,224, which is hereby incorporated by reference in its entirety.
[0075] In another aspect of this embodiment, an enterokinase is SEQ ID NO: 11. In another aspect of this embodiment, an enterokinase comprises amino acids 239-1035 of SEQ ID NO: 11. In yet another aspect of this embodiment, an enterokinase is a naturally occurring enterokinase variant, such as, e.g., an enterokinase isoform. In still another aspect of this embodiment, an enterokinase is a non-naturally occurring enterokinase variant, such as, e.g., a conservative enterokinase variant, a non-conservative enterokinase variant, an enterokinase chimeric, an active enterokinase fragment, or any combination thereof. In another aspect of this embodiment, an Enterokinase is one disclosed in U.S. Pat. No. 5,665,566 or U.S. Pat. No. 6,746,859. In another aspect of this embodiment, an enterokinase, a naturally occurring enterokinase variant, or a non-naturally occurring enterokinase variant is obtained from a species of mammal such as, e.g., a human, a cow, or a rodent.
[0076] In other aspects of this embodiment, an enterokinase comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 11; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 11. In yet other aspects of this embodiment, an enterokinase comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11. In still other aspects of this embodiment, an enterokinase comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 11.
[0077] In another aspect of this embodiment, a human rhinovirus 3C protease is SEQ ID NO: 12. In yet another aspect of this embodiment, a human rhinovirus 3C protease is a naturally occurring human rhinovirus 3C protease variant, such as, e.g., a human rhinovirus 3C protease isoform. In still another aspect of this embodiment, a human rhinovirus 3C protease is a non-naturally occurring human rhinovirus 3C protease variant, such as, e.g., a conservative human rhinovirus 3C protease variant, a non-conservative human rhinovirus 3C protease variant, a human rhinovirus 3C protease chimeric, an active human rhinovirus 3C protease fragment, or any combination thereof. In another aspect of this embodiment, a human rhinovirus 3C protease, a naturally occurring human rhinovirus 3C protease variant, or a non-naturally occurring human rhinovirus 3C protease variant is obtained from a species of Rhinovirus.
[0078] In other aspects of this embodiment, a human rhinovirus 3C protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 12; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 12. In yet other aspects of this embodiment, a human rhinovirus 3C protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12. In still other aspects of this embodiment, a human rhinovirus 3C protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 12.
[0079] In another aspect of this embodiment, a human enterovirus 3C protease is SEQ ID NO: 13. In yet another aspect of this embodiment, a human enterovirus 3C protease is a naturally occurring human enterovirus 3C protease variant, such as, e.g., a human enterovirus 3C protease isoform. In still another aspect of this embodiment, a human enterovirus 3C protease is a non-naturally occurring human enterovirus 3C protease variant, such as, e.g., a conservative human enterovirus 3C protease variant, a non-conservative human enterovirus 3C protease variant, a human enterovirus 3C protease chimeric, an active human enterovirus 3C protease fragment, or any combination thereof. In another aspect of this embodiment, a human enterovirus 3C protease, a naturally occurring human enterovirus 3C protease variant, or a non-naturally occurring human enterovirus 3C protease variant is obtained from a species of Enterovirus.
[0080] In other aspects of this embodiment, a human enterovirus 3C protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 13; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 13. In yet other aspects of this embodiment, a human enterovirus 3C protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13. In still other aspects of this embodiment, a human enterovirus 3C protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 13.
[0081] In another aspect of this embodiment, a TEV protease is SEQ ID NO: 14. In another aspect of this embodiment, a TEV protease comprises amino acids 2038-2270 of SEQ IS NO: 14. In another aspect of this embodiment, a TEV protease comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another aspect of this embodiment, a TEV protease is a naturally occurring TEV protease variant, such as, e.g., a TEV protease isoform. In still another aspect of this embodiment, a TEV protease is a non-naturally occurring TEV protease variant, such as, e.g., a conservative TEV protease variant, a non-conservative TEV protease variant, a TEV protease chimeric, an active TEV protease fragment, or any combination thereof. In another aspect of this embodiment, a TEV protease, a naturally occurring TEV protease variant, or a non-naturally occurring TEV protease variant is obtained from a species of Potyvirus.
[0082] In other aspects of this embodiment, a TEV protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet other aspects of this embodiment, a TEV protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In still other aspects of this embodiment, a TEV protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
[0083] In another aspect of this embodiment, a TVMV protease is SEQ ID NO: 24. In another aspect of this embodiment, a TEV protease comprises amino acids 2002-2236 of SEQ IS NO: 24. In yet another aspect of this embodiment, a TVMV protease is a naturally occurring TVMV protease variant, such as, e.g., a TVMV protease isoform. In still another aspect of this embodiment, a TVMV protease is a non-naturally occurring TVMV protease variant, such as, e.g., a conservative TVMV protease variant, a non-conservative TVMV protease variant, a TVMV protease chimeric, an active TVMV protease fragment, or any combination thereof. In another aspect of this embodiment, a TVMV protease, a naturally occurring TVMV protease variant, or a non-naturally occurring TVMV protease variant is obtained from a species of Potyvirus.
[0084] In other aspects of this embodiment, a TVMV protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24. In yet other aspects of this embodiment, a TVMV protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24. In still other aspects of this embodiment, a TVMV protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 24 or amino acids 2002-2236 of SEQ IS NO: 24.
[0085] In another aspect of this embodiment, a subtilisin protease is SEQ ID NO: 25. In another aspect of this embodiment, a subtilisin protease comprises amino acids 107-365 of SEQ IS NO: 25. In yet another aspect of this embodiment, a subtilisin protease is a naturally occurring subtilisin protease variant, such as, e.g., a subtilisin protease isoform. In still another aspect of this embodiment, a subtilisin protease is a non-naturally occurring subtilisin protease variant, such as, e.g., a conservative subtilisin protease variant, a non-conservative subtilisin protease variant, a subtilisin protease chimeric, an active subtilisin protease fragment, or any combination thereof. In another aspect of this embodiment, a subtilisin protease, a naturally occurring subtilisin protease variant, or a non-naturally occurring subtilisin protease variant is obtained from a species of Bacillus.
[0086] In other aspects of this embodiment, a subtilisin protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25. In yet other aspects of this embodiment, a subtilisin protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25. In still other aspects of this embodiment, a subtilisin protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 25 or amino acids 107-365 of SEQ IS NO: 25.
[0087] In another aspect of this embodiment, a Caspase 3 protease is SEQ ID NO: 26. In yet another aspect of this embodiment, a Caspase 3 protease is a naturally occurring Caspase 3 protease variant, such as, e.g., a Caspase 3 protease isoform. In still another aspect of this embodiment, a Caspase 3 protease is a non-naturally occurring Caspase 3 protease variant, such as, e.g., a conservative Caspase 3 protease variant, a non-conservative Caspase 3 protease variant, a Caspase 3 protease chimeric, an active Caspase 3 protease fragment, or any combination thereof. In another aspect of this embodiment, a Caspase 3 protease, a naturally occurring Caspase 3 protease variant, or a non-naturally occurring Caspase 3 protease variant is obtained from a species of mammal such as, e.g., a human, a cow, or a rodent.
[0088] In other aspects of this embodiment, a Caspase 3 protease comprises a polypeptide having an amino acid identity of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% to SEQ ID NO: 26; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or at most 95% to SEQ ID NO: 26. In yet other aspects of this embodiment, a Caspase 3 protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26. In still other aspects of this embodiment, a Caspase 3 protease comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions, and/or substitutions relative to SEQ ID NO: 26.
[0089] The methods disclosed in the present specification include, in part, a dual expression construct. A dual expression construct comprises two polynucleotide molecules, each including an open reading frame disclosed in the present specification operably-linked to an expression vector useful for expressing both polynucleotide molecules in a cell or cell-free extract. A wide variety of dual expression vectors can be employed for expressing a polynucleotide molecule disclosed in the present specification, including, without limitation, a viral dual expression vector; a prokaryotic dual expression vector; an eukaryotic dual expression vector, such as, e.g., a yeast dual expression vector, an insect dual expression vector and a mammalian dual expression vector; and a cell-free extract dual expression vector. It is further understood that dual expression vectors useful to practice aspects of these methods may include those which express the polynucleotide molecules under the control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. Non-limiting examples of dual expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, EMD Biosciences-Novagen, Madison, Wis. The selection, making and use of an appropriate dual expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.
[0090] The dual expression constructs disclosed in the present specification can comprise an open reading frame encoding a protein including a di-chain loop region comprising an exogenous protease cleavage site and another open reading frame encoding a protease that can cleave the exogenous protease cleavage site located within the di-chain loop, thereby converting the single-chain protein into its di-chain form.
[0091] Thus, in an embodiment, a dual expression construct comprises an open reading frame encoding a protein comprising a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification and another open reading frame encoding a protease that can cleave the exogenous protease cleavage site located within the di-chain loop as disclosed in the present specification.
[0092] In an aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a Clostridial toxin including a di-chain loop region comprising a TEV protease cleavage site and another open reading frame encoding a TEV protease. In another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a Clostridial toxin including a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site and another open reading frame encoding a TEV protease. In yet another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a Clostridial toxin including a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a Clostridial toxin binding domain, a di-chain loop region, and a TEV protease cleavage site, wherein the TEV protease cleavage site is located within the di-chain loop region and another open reading frame encoding a TEV protease.
[0093] In an aspect of this embodiment, a dual expression construct comprises an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising an exogenous protease cleavage site and another open reading frame encoding a protease that can cleave the exogenous protease cleavage site located within the di-chain loop region. In another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site and another open reading frame encoding a TEV protease. In yet another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, a di-chain loop region, and a TEV protease cleavage site, wherein the TEV protease cleavage site is located within the di-chain loop region and another open reading frame encoding a TEV protease.
[0094] In an aspect of this embodiment, a dual expression construct comprises an open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated protease cleavage site-binding domain. In another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, an integrated TEV protease cleavage site-binding domain and another open reading frame encoding a TEV protease. In yet another aspect of this embodiment, a dual expression construct can comprise one open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated TEV protease cleavage site-binding domain, wherein the TEV protease cleavage site is located within the di-chain loop region and another open reading frame encoding a TEV protease.
[0095] The location of one of the open reading frames contained within the dual expression construct can be in any order relative to the location of the other open reading frame, with the proviso that transcription from both open reading frames can still occur. When a dual expression construct is made, transcriptional initiation from the first promoter region typically transcribes both open reading frames, whereas, transcriptional initiation from the second promoter region typically transcribes only one of the open reading frames. Thus, depending on the location of the open reading frame relative to the first and second promoter regions, twice as many transcripts can be made from one of the open reading frames.
[0096] Thus, in one embodiment, the open reading frame encoding a protease is under the control of the first promoter region whereas the open reading frame encoding a protein comprising a di-chain loop region comprising an exogenous protease cleavage site is under the control of both the first promoter and second promoter regions. In an aspect of this embodiment, the open reading frame encoding a TEV protease is under the control of the first promoter region whereas the open reading frame encoding a Clostridial toxin comprising a TEV protease cleavage site located within the di-chain loop region is under the control of both the first promoter and second promoter regions. In another aspect of this embodiment, the open reading frame encoding a TEV protease is under the control of the first promoter region whereas the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site is under the control of both the first promoter and second promoter regions. In yet another aspect of this embodiment, the open reading frame encoding a TEV protease is under the control of the first promoter region whereas the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated TEV protease cleavage site-binding domain is under the control of both the first promoter and second promoter regions.
[0097] In another embodiment, the open reading frame encoding a protein comprising a di-chain loop region comprising an exogenous protease cleavage site is under the control of the first promoter region whereas the open reading frame encoding a protease is under the control of both the first promoter and second promoter regions. In an aspect of this embodiment, the open reading frame encoding a Clostridial toxin comprising a di-chain loop region comprising a TEV protease cleavage site is under the control of the first promoter region whereas the open reading frame encoding a TEV protease is under the control of both the first promoter and second promoter regions. In another aspect of this embodiment, the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a non-Clostridial toxin binding domain, and a di-chain loop region comprising a TEV protease cleavage site is under the control of the first promoter region whereas the open reading frame encoding a TEV protease is under the control of both the first promoter and second promoter regions. In yet another aspect of this embodiment, the open reading frame encoding a protein comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, and an integrated TEV protease cleavage site-binding domain is under the control of the first promoter region whereas the open reading frame encoding a TEV protease is under the control of both the first promoter and second promoter regions.
[0098] The 5'-3' orientation of one of the open reading frames contained within the dual expression construct can be in any direction relative to the 5'-3' orientation of the other open reading frame, with the proviso that transcription from both open reading frames can still occur. In one embodiment, the 5'-3' orientation of one of the open reading frames is in the same direction as the 5'-3' orientation of the other open reading frame. In another embodiment, the 5'-3' orientation of one of the open reading frames is in the opposite direction as the 5'-3' orientation of the other open reading frame. In an aspect of this embodiment, the 5'-3' orientation of one of the open reading frames is convergent relative to the 5'-3' orientation of the other open reading frame. In another aspect of this embodiment, the 5'-3' orientation of one of the open reading frames is divergent relative to the 5'-3' orientation of the other open reading frame.
[0099] The methods disclosed in the present specification include, in part, a protein comprising a di-chain loop region comprising an exogenous protease cleavage site. As used herein, the term "di-chain loop region" means the amino acid sequence of a Clostridial toxin containing a protease cleavage site used to convert the single-chain form of a Clostridial toxin into the di-chain form. Non-limiting examples of a Clostridial toxin di-chain loop region, include, a di-chain loop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1; a di-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; a di-chain loop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8; a di-chain loop region of BaNT comprising amino acids 421-435 of SEQ ID NO: 9; and a di-chain loop region of BuNT comprising amino acids 412-426 of SEQ ID NO: 10 (Table 2).
TABLE-US-00002 TABLE 2 Di-chain Loop Region of Clostridial Toxins Di-chain Loop Containing the Light Chain Naturally-occurring Protease Heavy Chain Toxin Region Cleavage Site Region BoNT/A NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*----ALNDLC IKVNNWDL BoNT/B KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGIC IDVDNEDL BoNT/C1 PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------TLDC RELLVKNTDL BoNT/D PALQKLSSESVVDLFTKV CLRLTKNSR*---------------DDSTC IKVKNNRL BoNT/E PRIITPITGRGLVKKIIRF CKNIVSVKGIR*--------------KSIC IEINNGEL BoNT/F PKIIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLC IRVNNSEL BoNT/G KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQC IIVNNEDL TeNT TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELC IKIKNEDL BaNT SRIVGPIPDNGLVERFVGL CKS-IVSKKGTK*------------NSLC IKVNNRDL BuNT PRIITPITGRGLVKKIIRF CKN-IVSVKGIR*--------------KSIC IEINNGEL The amino acid sequence displayed are as follows: BoNT/A, residues 410-462 of SEQ ID No: 1; BoNT/B, residues 418-454 of SEQ ID No: 2; BoNT/C1, residues 419-463 of SEQ ID No: 3; BoNT/D, residues 419-458 of SEQ ID No: 4; BoNT/E, residues 393-434 of SEQ ID No: 5; BoNT/F, residues 410-453 of SEQ ID No: 6; BoNT/G, residues 419-458 of SEQ ID No: 7; TeNT, residues 422-475 of SEQ ID No: 8; BaNT, residues 402-443 of SEQ ID No: 9; and BuNT, residues 393-434 of SEQ ID No: 10. An asterisks (*) indicates the peptide bond that is cleaved by a Clostridial toxin protease.
[0100] As mentioned above, Clostridial toxins are translated as a single-chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single disulphide bond and noncovalent interactions. While the identity of the protease is currently unknown, the di-chain loop protease cleavage site for many Clostridial toxins has been determined. In BoNTs, cleavage at K448-A449 converts the single polypeptide form of BoNT/A into the di-chain form; cleavage at K441-A442 converts the single polypeptide form of BoNT/B into the di-chain form; cleavage at K449-T450 converts the single polypeptide form of BoNT/C1 into the di-chain form; cleavage at R445-D446 converts the single polypeptide form of BoNT/D into the di-chain form; cleavage at R422-K423 converts the single polypeptide form of BoNT/E into the di-chain form; cleavage at K439-A440 converts the single polypeptide form of BoNT/F into the di-chain form; and cleavage at K446-S447 converts the single polypeptide form of BoNT/G into the di-chain form. Proteolytic cleavage of the single polypeptide form of TeNT at A457-5458 results in the di-chain form. Proteolytic cleavage of the single polypeptide form of BaNT at K431-N432 results in the di-chain form. Proteolytic cleavage of the single polypeptide form of BuNT at R422-K423 results in the di-chain form. Such a di-chain loop protease cleavage site is operably-linked in-frame to a modified Clostridial toxin as a fusion protein. However, it should also be noted that additional cleavage sites within the di-chain loop also appear to be cleaved resulting in the generation of a small peptide fragment being lost. As a non-limiting example, cleavage of a BoNT/A single-chain polypeptide ultimately results in the loss of a ten amino acid fragment within the di-chain loop.
[0101] It is envisioned that any molecule that comprises a di-chain loop region can be modified to include an exogenous protease cleavage site useful for the disclosed methods. Examples of molecules that can have the di-chain loop modified to include an exogenous protease cleavage site useful for the disclosed methods include, e.g., Keith A. Foster et al., Clostridial Toxin Derivatives Able To Modify Peripheral Sensory Afferent Functions, U.S. Pat. No. 5,989,545; Clifford C. Shone et al., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617; Conrad P. Quinn et al., Methods and Compounds for the Treatment of Mucus Hypersecretion, U.S. Pat. No. 6,632,440; Lance E. Steward et al., Methods And Compositions For The Treatment Of Pancreatitis, U.S. Pat. No. 6,843,998; Stephan Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,244,437; Stephan Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,413,742; Stephan Donovan, Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. Pat. No. 7,425,338; each of which is hereby incorporated by reference in its entirety.
[0102] A di-chain loop region is modified by the addition of an exogenous protease cleavage site. As used herein, the term "exogenous protease cleavage site" is synonymous with a "non-naturally occurring protease cleavage site" or "non-native protease cleavage site" and refers to a protease cleavage site that is not normally present in a di-chain loop region from a naturally occurring Clostridial toxin. It is envisioned that any and all exogenous protease cleavage sites that can be used to convert the single-chain polypeptide form of a Clostridial toxin into the di-chain form are useful to practice aspects of the present invention. Non-limiting examples of exogenous protease cleavage sites include, e.g., an enterokinase protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a tobacco etch virus (TEV) protease cleavage site, a Tobacco Vein Mottling Virus (TVMV) protease cleavage site, a subtilisin protease cleavage site, or a Caspase 3 protease cleavage site.
[0103] It is envisioned that an exogenous protease cleavage site of any and all lengths can be useful in aspects of the present invention with the proviso that the exogenous protease cleavage site is capable of being cleaved by its respective protease. Thus, in aspects of this embodiment, an exogenous protease cleavage site can have a length of, e.g., at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 amino acids; or at most 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 amino acids.
[0104] In an embodiment, a di-chain loop region comprises an exogenous protease cleavage site. In aspects of this embodiment, a di-chain loop region is modified to comprise, e.g., an enterokinase protease cleavage site, a Tobacco Etch Virus protease cleavage site, a Tobacco Vein Mottling Virus protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a subtilisin cleavage site, and a Caspase 3 cleavage site. In other aspects of this embodiment, an exogenous protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, an exogenous protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0105] In an aspect of this embodiment, a di-chain loop region comprises a Tobacco Etch Virus protease cleavage site having the consensus sequence E-P5-P4-Y--P2-Q*-G (SEQ ID NO: 27) or E-P5-P4-Y--P2-Q*-S (SEQ ID NO: 28), where P2, P4 and P5 can be any amino acid. In other aspects of the embodiment, a di-chain loop region comprises a Tobacco Etch Virus protease cleavage site comprising SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38. In still other aspects of this embodiment, a Tobacco Etch Virus protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a Tobacco Etch Virus protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0106] In another aspect of this embodiment, a di-chain loop region comprises a Tobacco Vein Mottling Virus protease cleavage site having the consensus sequence P6-P5-V--R--F-Q*-G (SEQ ID NO: 39) or P6-P5-V--R--F-Q*-S (SEQ ID NO: 40), where P5 and P6 can be any amino acid. In other aspects of the embodiment, a di-chain loop region comprises a Tobacco Vein Mottling Virus protease cleavage site comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44. In still other aspects of this embodiment, a Tobacco Vein Mottling Virus protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a Tobacco Vein Mottling Virus protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0107] In yet another aspect of this embodiment, a di-chain loop region comprises a human rhinovirus 3C protease cleavage site having the consensus sequence P5-P4-L-F-Q*-G-P (SEQ ID NO: 45), where P4 is G, A, V, L, I, M, S or T and P5 can any amino acid, with D or E preferred. In other aspects of the embodiment, a di-chain loop region comprises a human rhinovirus 3C protease cleavage site comprising SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In still other aspects of this embodiment, a human rhinovirus 3C protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a human rhinovirus 3C protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0108] In still another aspect of this embodiment, a di-chain loop region comprises a subtilisin protease cleavage site having the consensus sequence P6-P5-P4-P3-H*-Y (SEQ ID NO: 52) or P6-P5-P4-P3-Y--H* (SEQ ID NO: 53), where P3, P4 and P5 and P6 can be any amino acid. In other aspects of the embodiment, a di-chain loop region comprises a subtilisin protease cleavage site comprising SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. In still other aspects of this embodiment, a subtilisin protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a subtilisin protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0109] In a further aspect of this embodiment, a di-chain loop region comprises a Caspase 3 protease cleavage site having the consensus sequence D-P3-P2-D*P1' (SEQ ID NO: 57), where P3 can be any amino acid, with E preferred, P2 can be any amino acid and P1' can any amino acid, with G or S preferred. In other aspects of the embodiment, a di-chain loop region comprises a Caspase 3 protease cleavage site comprising SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In still other aspects of this embodiment, a Caspase 3 protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In other aspects of this embodiment, a Caspase 3 protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0110] In yet another aspect of this embodiment, a di-chain loop region comprises an enterokinase protease cleavage site having the consensus sequence DDDDK (SEQ ID NO: 64). In other aspects of this embodiment, an enterokinase protease cleavage site is located within the di-chain loop of, e.g., a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. In yet other aspects of this embodiment, an enterokinase protease cleavage site is located within the di-chain loop of a protein disclosed in, e.g., U.S. Pat. No. 5,989,545; U.S. Pat. No. 6,461,617; U.S. Pat. No. 6,632,440; U.S. Pat. No. 6,843,998; U.S. Pat. No. 7,244,437; U.S. Pat. No. 7,413,742; and U.S. Pat. No. 7,425,338.
[0111] A di-chain loop region is modified to replace a naturally-occurring di-chain loop protease cleavage site for an exogenous protease cleavage site. In this modification, the naturally-occurring di-chain loop protease cleavage site is made inoperable and thus can not be cleaved by its protease. Only the exogenous protease cleavage site can be cleaved by its corresponding exogenous protease. In this type of modification, the exogenous protease site is operably-linked in-frame to a modified Clostridial toxin as a fusion protein and the site can be cleaved by its respective exogenous protease. Replacement of an endogenous di-chain loop protease cleavage site with an exogenous protease cleavage site can be a substitution of the sites where the exogenous site is engineered at the position approximating the cleavage site location of the endogenous site. Replacement of an endogenous di-chain loop protease cleavage site with an exogenous protease cleavage site can be the addition of an exogenous site where the exogenous site is engineered at a position different from the cleavage site location of the endogenous site, the endogenous site being engineered to be inoperable.
[0112] A naturally-occurring protease cleavage site contained within the di-chain loop region can be made inoperable by altering at least the two amino acids flanking the peptide bond cleaved by the naturally-occurring di-chain loop protease. More extensive alterations can be made, with the proviso that the two cysteine residues of the di-chain loop region remain intact and the region can still form a disulfide bridge. Non-limiting examples of an amino acid alteration include deletion of an amino acid or replacement of the original amino acid with a different amino acid. Thus, in one embodiment, a naturally-occurring protease cleavage site contained within the di-chain loop region is made inoperable by altering the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease. In other aspects of this embodiment, a naturally-occurring protease cleavage site contained within the di-chain loop region is made inoperable by altering, e.g., at least three amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least four amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least five amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least six amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least seven amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least eight amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least nine amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least ten amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at least 15 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; or at least 20 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease.
[0113] In still other aspects of this embodiment, a naturally-occurring di-chain protease cleavage site contained within the di-chain loop region is made inoperable by altering, e.g., at most three amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most four amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most five amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most six amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most seven amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most eight amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most nine amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most ten amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; at most 15 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease; or at most 20 amino acids including the two amino acids flanking the peptide bond cleaved by a naturally-occurring protease.
[0114] The methods disclosed in the present specification include, in part, a cell. It is envisioned that any and all cells can be used. Thus, aspects of this embodiment include, without limitation, prokaryotic cells including, without limitation, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells such as those derived from, e.g., Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile, Caulobacter crescentus, Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls, Neisseria meningitidis, Pseudomonas fluorescens and Salmonella typhimurium; and eukaryotic cells including, without limitation, yeast strains, such as, e.g., those derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as, e.g., those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca sexta; and mammalian cells and cell lines derived from mammalian cells, such as, e.g., those derived from mouse, rat, hamster, porcine, bovine, equine, primate and human. Cell lines may be obtained from the American Type Culture Collection, European Collection of Cell Cultures and the German Collection of Microorganisms and Cell Cultures. Non-limiting examples of specific protocols for selecting, making and using an appropriate cell line are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F. A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer Academic Publishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, 4th ed. 2000); ANIMAL CELL CULTURE: A PRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press, 3rd ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2nd ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.
[0115] The methods disclosed in the present specification include, in part, introducing into a cell an expression construct or dual expression construct as disclosed in the present specification. An expression construct or dual expression construct introduced into a cell can be transiently or stably maintained by that cell. Stably-maintained expression constructs or dual expression constructs may be extra-chromosomal and replicate autonomously, or they may be integrated into the chromosomal material of the cell and replicate non-autonomously. It is envisioned that any and all methods for introducing an expression construct or a dual expression construct disclosed in the present specification into a cell can be used. Methods useful for introducing an expression construct or a dual expression construct into a cell include, without limitation, chemical-mediated transfection such as, e.g., calcium phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediated and polybrene-mediated; physical-mediated tranfection, such as, e.g., biolistic particle delivery, microinjection, protoplast fusion and electroporation; and viral-mediated transfection, such as, e.g., retroviral-mediated transfection, see, e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3rd ed. 2001). One skilled in the art understands that selection of a specific method to introduce an expression construct or a dual expression construct into a cell will depend, in part, on whether the cell will transiently contain the expression construct or dual expression construct, or whether the cell will stably contain the expression construct or dual expression construct. These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.
[0116] In an aspect of this embodiment, a chemical-mediated method, termed transfection, is used to introduce an expression construct or a dual expression construct disclosed in the present specification into a cell. In chemical-mediated methods of transfection the chemical reagent forms a complex with the expression construct or dual expression construct that facilitates its uptake into the cells. Such chemical reagents include, without limitation, calcium phosphate-mediated, see, e.g., Martin Jordan & Florian Worm, Transfection of adherent and suspended cells by calcium phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationic polymer-mediated like polyethyleneimine (PEI)-mediated and polylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine strategies for plasmid delivery to brain-derived cells, 33(2) Methods 144-150 (2004). Such chemical-mediated delivery systems can be prepared by standard methods and are commercially available, see, e.g., CellPhect Transfection Kit (Amersham Biosciences, Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine® Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500 Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect and Effectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).
[0117] In another aspect of this embodiment, a physically-mediated method is used to introduce an expression construct or a dual expression construct disclosed in the present specification into a cell. Physical techniques include, without limitation, electroporation, biolistic and microinjection. Biolistics and microinjection techniques perforate the cell wall in order to introduce the expression construct or dual expression construct into the cell, see, e.g., Jeike E. Biewenga et al., Plasmid-mediated gene transfer in neurons using the biolistics technique, 71(1) J. Neurosci. Methods. 67-75 (1997); and John O'Brien & Sarah C. R. Lummis, Biolistic and diolistic transfection: using the gene gun to deliver DNA and lipophilic dyes into mammalian cells, 33(2) Methods 121-125 (2004). Electroporation, also termed electropermeabilization, uses brief, high-voltage, electrical pulses to create transient pores in the membrane through which the polynucleotide molecules enter and can be used effectively for stable and transient transfections of all cell types, see, e.g., M. Golzio et al., In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression, 33(2) Methods 126-135 (2004); and Oliver Greschet al., New non-viral method for gene transfer into primary cells, 33(2) Methods 151-163 (2004).
[0118] In another aspect of this embodiment, a viral-mediated method, termed transduction, is used to introduce an expression construct or a dual expression construct disclosed in the present specification into a cell. In viral-mediated methods of transient transduction, the process by which viral particles infect and replicate in a host cell has been manipulated in order to use this mechanism to introduce the expression construct or dual expression construct into the cell. Viral-mediated methods have been developed from a wide variety of viruses including, without limitation, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); and Maurizio Federico, From lentiviruses to lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M. Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther. 529-540 (2003); Karim Benihoud et al, Adenovirus vectors for gene delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler, Adeno-associated viral vectors for gene transfer and gene therapy, 380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al., Adenovirus and adeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002); Edward A. Burton et al., Gene delivery using herpes simplex virus vectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al., Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); Ilya Frolov et al., Alphavirus-based expression vectors: strategies and applications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996); Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1) Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay, Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4) Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann, Baculovirus vectors: novel mammalian cell gene-delivery vehicles and their applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003).
[0119] Adenoviruses, which are non-enveloped, double-stranded DNA viruses, are often selected for mammalian cell transduction because adenoviruses handle relatively large polynucleotide molecules of about 36 kb, are produced at high titer, and can efficiently infect a wide variety of both dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermens et al., Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci. Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches for generating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev. 165-176 (2001). Transduction using adenoviral-based system do not support prolonged protein expression because the nucleic acid molecule is carried by an episome in the cell nucleus, rather than being integrated into the host cell chromosome. Adenoviral vector systems and specific protocols for how to use such vectors are disclosed in, e.g., VIRAPOWER® Adenoviral Expression System (Invitrogen, Inc., Carlsbad, Calif.) and VIRAPOWER® Adenoviral Expression System Instruction Manual 25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and ADEASY® Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) and ADEASY® Adenoviral Vector System Instruction Manual 064004f, Stratagene, Inc.
[0120] Introduction of an expression construct or dual expression construct disclosed in the present specification into a cell can also be achieved using single-stranded RNA retroviruses, such as, e.g., oncoretroviruses and lentiviruses. Retroviral-mediated transduction often produce transduction efficiencies close to 100%, can easily control the proviral copy number by varying the multiplicity of infection (MOI), and can be used to either transiently or stably transduce cells, see, e.g., Tiziana Tonini et al., Transient production of retroviral-and lentiviral-based vectors for the transduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004); Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); Felix Recillas-Targa, Gene transfer and expression in mammalian cell lines and transgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and Roland Wolkowicz et al., Lentiviral vectors for the delivery of DNA into mammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviral particles consist of an RNA genome packaged in a protein capsid, surrounded by a lipid envelope. The retrovirus infects a host cell by injecting its RNA into the cytoplasm along with the reverse transcriptase enzyme. The RNA template is then reverse transcribed into a linear, double stranded cDNA that replicates itself by integrating into the host cell genome. Viral particles are spread both vertically (from parent cell to daughter cells via the provirus) as well as horizontally (from cell to cell via virions). This replication strategy enables long-term persistent expression since the nucleic acid molecules of interest are stably integrated into a chromosome of the host cell, thereby enabling long-term expression of the protein. For instance, animal studies have shown that lentiviral vectors injected into a variety of tissues produced sustained protein expression for more than 1 year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector, 272(5259) Science 263-267 (1996). The Oncoretroviruses-derived vector systems, such as, e.g., Moloney murine leukemia virus (MoMLV), are widely used and infect many different non-dividing cells. Lentiviruses can also infect many different cell types, including dividing and non-dividing cells and possess complex envelope proteins, which allows for highly specific cellular targeting.
[0121] Retroviral vectors and specific protocols for how to use such vectors are disclosed in, e.g., Manfred Gossen & Hermann Bujard, Tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters, U.S. Pat. No. 5,464,758, Hermann Bujard & Manfred Gossen, Methods for regulating gene expression, U.S. Pat. No. 5,814,618, David S. Hogness, Polynucleotides encoding insect steroid hormone receptor polypeptides and cells transformed with same, U.S. Pat. No. 5,514,578, and David S. Hogness, Polynucleotide encoding insect ecdysone receptor, U.S. Pat. No. 6,245,531; Elisabetta Vegeto et al., Progesterone receptor having C. terminal hormone binding domain truncations, U.S. Pat. No. 5,364,791, Elisabetta Vegeto et al., Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy, U.S. Pat. No. 5,874,534, and Elisabetta Vegeto et al., Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy, U.S. Pat. No. 5,935,934. Furthermore, such viral delivery systems can be prepared by standard methods and are commercially available, see, e.g., BD® Tet-Off and Tet-On Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto, Calif.) and BD® Tet-Off and Tet-On Gene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech, (Mar. 14, 2003), GENESWITCH® System (Invitrogen, Inc., Carlsbad, Calif.) and GENESWITCH® System A Mifepristone-Regulated Expression System for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002); VIRAPOWER® Lentiviral Expression System (Invitrogen, Inc., Carlsbad, Calif.) and VIRAPOWER® Lentiviral Expression System Instruction Manual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and COMPLETE CONTROL® Retroviral Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.) and COMPLETE CONTROL® Retroviral Inducible Mammalian Expression System Instruction Manual, 064005e.
[0122] The methods disclosed in the present specification include, in part, expressing an expression construct or dual expression construct disclosed in the present specification. It is envisioned that any of a variety of expression systems may be useful for expressing an expression construct or a dual expression construct disclosed in the present specification, including, without limitation, cell-based systems, and cell-free expression systems. Cell-based systems include, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Cell-free systems include, without limitation, wheat germ extracts, rabbit reticulocyte extracts and E. coli extracts and generally are equivalent to the method disclosed herein. Expression of an expression construct or dual expression construct using an expression system can include any of a variety of characteristics including, without limitation, inducible expression, non-inducible expression, constitutive expression, viral-mediated expression, stably-integrated expression, and transient expression. Expression systems that include well-characterized vectors, reagents, conditions and cells are well-established and are readily available from commercial vendors that include, without limitation, Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science, Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limiting examples on the selection and use of appropriate heterologous expression systems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Hames eds., Oxford University Press, 1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai & Harish Padh, Expression Systems for Production of Heterologous Proteins, 80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.
[0123] A variety of cell-based expression procedures are useful for expressing an expression construct or a dual expression construct disclosed in the present specification. Examples included, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Viral expression systems include, without limitation, the VIRAPOWER® Lentiviral (Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral Expression Systems (Invitrogen, Inc., Carlsbad, Calif.), the ADEASY® XL Adenoviral Vector System (Stratagene, La Jolla, Calif.) and the VIRAPORT® Retroviral Gene Expression System (Stratagene, La Jolla, Calif.). Non-limiting examples of prokaryotic expression systems include the CHAMPION® pET Expression System (EMD Biosciences-Novagen, Madison, Wis.), the TRIEX® Bacterial Expression System (EMD Biosciences-Novagen, Madison, Wis.), the QIAEXPRESS® Expression System (QIAGEN, Inc.), and the AFFINITY® Protein Expression and Purification System (Stratagene, La Jolla, Calif.). Yeast expression systems include, without limitation, the EASYSELECT® Pichia Expression Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-ECHO® Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and the SPECTRA® S. pombe Expression System (Invitrogen, Inc., Carlsbad, Calif.). Non-limiting examples of baculoviral expression systems include the BACULODIRECT® (Invitrogen, Inc., Carlsbad, Calif.), the BAC-TO-BAC® (Invitrogen, Inc., Carlsbad, Calif.), and the BD BACULOGOLD® (BD Biosciences-Pharmigen, San Diego, Calif.). Insect expression systems include, without limitation, the Drosophila Expression System (DES®) (Invitrogen, Inc., Carlsbad, Calif.), INSECTSELECT® System (Invitrogen, Inc., Carlsbad, Calif.) and INSECTDIRECT® System (EMD Biosciences-Novagen, Madison, Wis.). Non-limiting examples of mammalian expression systems include the T-REXT® (Tetracycline-Regulated Expression) System (Invitrogen, Inc., Carlsbad, Calif.), the FLP-IN® T-REX® System (Invitrogen, Inc., Carlsbad, Calif.), the pcDNA® system (Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2 system (Invitrogen, Inc., Carlsbad, Calif.), the EXCHANGER® System, INTERPLAY® Mammalian TAP System (Stratagene, La Jolla, Calif.), COMPLETE CONTROL® Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.) and LACSWITCH® II Inducible Mammalian Expression System (Stratagene, La Jolla, Calif.).
[0124] Another procedure of expressing an expression construct or a dual expression construct disclosed in the present specification employs a cell-free expression system such as, without limitation, prokaryotic extracts and eukaryotic extracts. Non-limiting examples of prokaryotic cell extracts include the RTS 100 E. coli HY Kit (Roche Applied Science, Indianapolis, Ind.), the ACTIVEPRO® In Vitro Translation Kit (Ambion, Inc., Austin, Tex.), the ECOPRO® System (EMD Biosciences-Novagen, Madison, Wis.) and the Expressway® Plus Expression System (Invitrogen, Inc., Carlsbad, Calif.). Eukaryotic cell extract include, without limitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science, Indianapolis, Ind.), the TNT® Coupled Wheat Germ Extract Systems (Promega Corp., Madison, Wis.), the Wheat Germ IVT® Kit (Ambion, Inc., Austin, Tex.), the Retic Lysate IVT® Kit (Ambion, Inc., Austin, Tex.), the PROTEINSCRIPT®.sup. II System (Ambion, Inc., Austin, Tex.) and the TNT®.sup. Coupled Reticulocyte Lysate Systems (Promega Corp., Madison, Wis.).
[0125] The methods disclosed in the present specification include, in part, growing a cell at a first temperature for a certain period of time and then growing the cell at a second temperature for a certain period of time. The first and second temperatures and the periods of time the cells are grown at the first and second temperatures are determined based on the desired amount of protein to be expressed by the cell, and the desired cleavage efficiency at the exogenous protease cleavage site located within the di-chain loop region to convert the single-chain protein into its di-chain form.
[0126] In one embodiment, a cell is grown at a first temperature for a certain period of time in order to achieve maximum cell density. In aspects of this embodiment, a cell is grown at about 37° C. for about 0.5 hours, about 1.0 hour, about 1.5 hours, about 2.0 hours, about 3.0 hours, about 3.5 hours, about 4.0 hours, about 5.0 hours, about 6.0 hours, about 7.0 hours, about 8.0 hours, about 9.0 hours or about 10 hours. In other aspects of this embodiment, a cell is grown at about 42° C. for about 0.5 hours, about 1.0 hour, about 1.5 hours, about 2.0 hours, about 3.0 hours, about 3.5 hours, about 4.0 hours, about 5.0 hours. In aspects of this embodiment, a cell is grown at about 30° C. for about 0.5 hours, about 1.0 hour, about 1.5 hours, about 2.0 hours, about 3.0 hours, about 3.5 hours, about 4.0 hours, or about 5.0 hours. In yet other aspects, of this embodiment, a cell is grown at about 12° C. for about 2 hours to about 8 hours, at about 16° C. for about 2 hours to about 8 hours, at about 20° C. for about 2 hours to about 8 hours, or at about 24° C. for about 2 hours to about 8 hours. In still other aspects, of this embodiment, a cell is grown at about 12° C. to about 16° C. for about 2 hours to about 8 hours, or at about 20° C. to about 24° C. for about 2 hours to about 8 hours.
[0127] In another embodiment, a cell is grown at a second temperature for a certain period of time in order to achieve maximum induction of protein expression. In aspects of this embodiment, a cell is grown at about 37° C. for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In other aspects of this embodiment, a cell is grown at about 30° C. for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In yet other aspects of this embodiment, a cell is grown at about 25° C. for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In still other aspects of this embodiment, a cell is grown at about 22° C. for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In further aspects of this embodiment, a cell is grown at about 16° C. for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours. In yet further aspects of this embodiment, a cell is grown at about 12° C. for about 1.5 hours, about 2.5 hours, about 3.5 hours, about 4.5 hours, about 5.5 hours, about 6.5 hours, about 7.5 hours, about 8.5 hours, about 9.5 hours, about 10.5 hours, about 11.5 hours, about 12.5 hours, about 13.5 hours, about 14.5 hours, about 15.5 hours, about 16.5 hours, or about 24.5 hours.
[0128] Aspects of the present invention can also be described as follows:
[0129] 1. An intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of:
[0130] a) growing a cell comprising a dual expression construct at a first temperature for a certain period of time in order to achieve maximal cell density, the dual expression construct comprising;
[0131] i) an open reading frame encoding a single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; and
[0132] ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop;
[0133] b) growing the cell at a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein, wherein growth at step (b) induces expression of the single-chain protein and the protease from the dual expression construct; and
[0134] wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0135] 2. An intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of:
[0136] a) growing a cell comprising a dual expression construct at 37° C. for about 3.5 hours, the dual expression construct comprising;
[0137] i) an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; and
[0138] ii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop;
[0139] b) growing the cell at 22° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain Clostridial toxin and the protease from the dual expression construct; and
[0140] wherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.
[0141] 3. The intracellular method according to 2, wherein the single-chain Clostridial toxin comprises a linear amino-to-carboxyl single polypeptide order of 1) the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin translocation domain and the Clostridial toxin binding domain; 2) the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin binding domain and the Clostridial toxin translocation domain; 3) the Clostridial toxin binding domain, the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin enzymatic domain; 4) the Clostridial toxin binding domain, the Clostridial toxin enzymatic domain, the di-chain loop region comprising an exogenous protease cleavage site and the Clostridial toxin translocation domain; 5) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial toxin enzymatic domain and the Clostridial toxin binding domain; or 6) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the Clostridial binding domain and the Clostridial toxin enzymatic domain.
[0142] 4. The intracellular method according to 2, wherein the Clostridial toxin enzymatic domain is a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain, or a BuNT enzymatic domain.
[0143] 5. The intracellular method according to 2, wherein the Clostridial toxin translocation domain is a BoNT/A translocation domain, a BoNT/B translocation domain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, a BoNT/E translocation domain, a BoNT/F translocation domain, a BoNT/G translocation domain, a TeNT translocation domain, a BaNT translocation domain, or a BuNT translocation domain.
[0144] 6. The intracellular method according to 2, wherein the Clostridial toxin binding domain is a BoNT/A binding domain, a BoNT/B binding domain, a BoNT/C1 binding domain, a BoNT/D binding domain, a BoNT/E binding domain, a BoNT/F binding domain, a BoNT/G binding domain, a TeNT binding domain, a BaNT binding domain, or a BuNT binding domain.
[0145] 7. The intracellular method according to 2, wherein the exogenous protease cleavage site is an enterokinase protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a tobacco etch virus (TEV) protease cleavage site, a Tobacco Vein Mottling Virus (TVMV) protease cleavage site, a subtilisin protease cleavage site, or a Caspase 3 protease cleavage site.
[0146] 8. The intracellular method according to 2, wherein the protease is an enterokinase protease, a human rhinovirus 3C protease, a human enterovirus 3C protease, a tobacco etch virus (TEV) protease, a Tobacco Vein Mottling Virus (TVMV) protease, a subtilisin protease, or a Caspase 3 protease.
[0147] 9. An intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of
[0148] a) growing a cell comprising a dual expression construct at 37° C. for about 8 hours, the dual expression construct comprising;
[0149] i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, an integrated TEV protease cleavage site-opioid binding domain; and
[0150] ii) an open reading frame encoding a TEV protease;
[0151] b) growing the cell at about 12 to about 16° C. for about 16 to about 18 hours,
[0152] wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and
[0153] wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the integrated TEV cleavage site opioid binding domain, thereby converting the single-chain protein into its di-chain form.
[0154] 10. The intracellular method according to 9, wherein the protein comprises a linear amino-to-carboxyl single polypeptide order of 1) the Clostridial toxin enzymatic domain, the Clostridial toxin translocation domain, and the integrated TEV protease cleavage site-opioid binding domain, 2) the Clostridial toxin enzymatic domain, the integrated TEV protease cleavage site-opioid binding domain, and the Clostridial toxin translocation domain, 3) the integrated TEV protease cleavage site-opioid binding domain, the Clostridial toxin translocation domain, and the Clostridial toxin enzymatic domain, 4) the integrated TEV protease cleavage site-opioid binding domain, the Clostridial toxin enzymatic domain, and the Clostridial toxin translocation domain, 5) the Clostridial toxin translocation domain, the integrated TEV protease cleavage site-opioid binding domain, and the Clostridial toxin enzymatic domain, or 6) the Clostridial toxin translocation domain, the Clostridial toxin enzymatic domain, and the integrated TEV protease cleavage site-opioid binding domain.
[0155] 11. The intracellular method according to 9, wherein the Clostridial toxin enzymatic domain is a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain, or a BuNT enzymatic domain.
[0156] 12. The intracellular method according to 9, wherein the Clostridial toxin translocation domain is a BoNT/A translocation domain, a BoNT/B translocation domain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, a BoNT/E translocation domain, a BoNT/F translocation domain, a BoNT/G translocation domain, a TeNT translocation domain, a BaNT translocation domain, or a BuNT translocation domain.
[0157] 13. The intracellular method according to 9, wherein the integrated TEV protease cleavage site-opiod binding domain is an integrated TEV protease cleavage site-nociceptin binding domain, an integrated TEV protease cleavage site-dynorphin binding domain, an integrated TEV protease cleavage site-enkephalin binding domain, an integrated TEV protease cleavage site-BAM22 binding domain, an integrated TEV protease cleavage site-endomorphin binding domain, an integrated TEV protease cleavage site-endorphin binding domain, an integrated TEV protease cleavage site-hemorphin binding domain, or an integrated TEV protease cleavage site-rimorphin binding domain.
[0158] 14. An intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of
[0159] a) growing a cell comprising a dual expression construct at 37° C. for about 8 hours, the dual expression construct comprising;
[0160] i) an open reading frame encoding a single-chain protein, the single-chain protein comprising an enzymatic domain, a translocation domain, a non-Clostridial toxin binding domain and a di-chain loop region comprising a TEV protease cleavage site; and
[0161] ii) an open reading frame encoding a TEV protease;
[0162] b) growing the cell at about 12 to about 16° C. for about 16 to about 18 hours, wherein growth at step (b) induces expression of the single-chain protein and the TEV protease from the dual expression construct; and
[0163] wherein the produced TEV protease cleaves the single-chain protein at the TEV protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
[0164] 15. The intracellular method according to 14, wherein the single-chain Clostridial toxin comprises a linear amino-to-carboxyl single polypeptide order of 1) the Clostridial toxin enzymatic domain, the di-chain loop region comprising a TEV protease cleavage site, the Clostridial toxin translocation domain and the non-Clostridial toxin binding domain; 2) the Clostridial toxin enzymatic domain, the di-chain loop region comprising a TEV protease cleavage site, the non-Clostridial toxin binding domain and the Clostridial toxin translocation domain; 3) the non-Clostridial toxin binding domain, the Clostridial toxin translocation domain, the di-chain loop region comprising a TEV protease cleavage site and the Clostridial toxin enzymatic domain; 4) the non-Clostridial toxin binding domain, the Clostridial toxin enzymatic domain, the di-chain loop region comprising a TEV protease cleavage site and the Clostridial toxin translocation domain; 5) the Clostridial toxin translocation domain, the di-chain loop region comprising a TEV protease cleavage site, the Clostridial toxin enzymatic domain and the non-Clostridial toxin binding domain; or 6) the Clostridial toxin translocation domain, the di-chain loop region comprising an exogenous protease cleavage site, the non-Clostridial binding domain and the Clostridial toxin enzymatic domain.
[0165] 16. The intracellular method according to 14, wherein the Clostridial toxin enzymatic domain is a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain, or a BuNT enzymatic domain.
[0166] 17. The intracellular method according to 14, wherein the Clostridial toxin translocation domain is a BoNT/A translocation domain, a BoNT/B translocation domain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, a BoNT/E translocation domain, a BoNT/F translocation domain, a BoNT/G translocation domain, a TeNT translocation domain, a BaNT translocation domain, or a BuNT translocation domain.
[0167] 18. The intracellular method according to 14, wherein the non-Clostridial toxin binding domain is an opioid peptide binding domain, a melanocortin peptide binding domain, a galanin peptide binding domain, a granin peptide binding domain, a tachykinin peptide binding domain, a neuropeptide Y related peptide binding domain, a neurohormone peptide binding domain, a cytokine peptide binding domain, a kinin peptide binding domain, a fibroblast growth factor peptide binding domain, a neurotrophin peptide binding domain, a tumor necrosis factor peptide binding domain, a glial derived neurotrophic factor peptide binding domain, a transformation growth factor 6 peptide binding domain, a bone morphogenetic protein peptide binding domain, a growth and differentiation factor peptide binding domain, an activin peptide binding domain, a vascular endothelial growth factor peptide binding domain, an insulin growth factor peptide binding domain, an epidermial growth factor peptide binding domain, a glucagon like hormone peptide binding domain, a pituitary adenylate cyclase activating peptide binding domain, a growth hormone-releasing hormone peptide binding domain, a vasoactive intestinal peptide binding domain, a gastric inhibitory polypeptide peptide binding domain, a calcitonin-related peptidesvisceral gut peptide binding domain, or a protease activated receptor peptide binding domain.
EXAMPLES
Example 1
TEV Protease Variants
[0168] The following example illustrates how to make and use TEV protease variants that have increased stability and/or solubility.
A. Construction of pET29/TEV Expression Constructs.
[0169] In order to produce a TEV protease recombinantly, an open reading frame encoding the desired TEV protease was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, Wash.). Complementary oligonucleotides of 20 to 50 bases in length, spanning the entire open reading frame, were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHB1 carrier vector at the SmaI site to generate pUCBHB1/TEV plasmids. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR® Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).
[0170] The open reading frame encoding the TEV variants were codon-optimized for E. coli expression and all encode an approximately 250 amino acid proteolytic fragment of approximately 27.5 kDa, corresponding to residues 2038-2279 of the full-length TEV polyprotein fused to either an N- or C-terminal poly-histidine affinity purification tag. Recombinant expression of wild-type TEV protease results in a protein that has a propensity to cleave itself at Serine 219 to generate a truncated protease with greatly diminished proteolytic activity. Thus, to largely eliminate autoproteolysis and subsequent generation of this truncated product, TEV variants were synthesized where Serine 219 was changed to either Asparagine (S219N) or Valine (S219V). In addition, it is well documented that although recombinant wild-type TEV protease is expressed at very high levels in E. coli, it is almost entirely insoluble (Kapust et al., 2001). Thus, to improve solubility of the expressed TEV, several amino acid variants were made and tested to determine whether the changes resulted in increased protein solubility. The TEV variants synthesized are shown in Table 3. Variant 1 represented a codon-optimized TEV construct engineered with a C-terminal His-tag and the S219N mutation. Variant 11 was a construct with native DNA sequence of TEV protease engineered with an N-terminal tag and the S219N mutation.
TABLE-US-00003 TABLE 3 TEV Protease Variants Autoproteolysis DNA Protein Elimination Affinity SEQ ID SEQ ID Variant Change Solubility Enhancing Changes Tag NO: NO: 1 S219N -- C-term 65 66 2 S219N L56V, S135G N-term 67 68 3 S219N T17S, N68D, I77V N-term 69 70 4 S219N N44V, L56V, S135G N-term 71 72 5 S219N L56V, N68D, S135G N-term 73 74 6 S219N T17S, L56V, N68D, I77V N-term 75 76 7 S219N T17S, N68D, I77V, S135G N-term 77 78 8 S219N T17S, N44V, L56V, N68D, I77V, S135G C-term 79 80 9 S219V T17S, N44V, L56V, N68D, I77V, S135G N-term 81 82 10 S219N T17S, N44V, L56V, N68D, I77V, S135G N-term 83 84 11 S219N -- N-term 85 86
[0171] To construct pET29/TEV variant expression constructs, a pUCBHB1/TEV construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding the TEV; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). Using a T4 DNA ligase procedure this insert was directionally ligated into a pET29 vector digested with the same restriction endonucleases in the multiple cloning site. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the TEV gene insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding TEV variants operably-linked to either a carboxyl terminal or amino-terminal polyhistidine affinity purification peptide.
B. Analysis of TEV Expression Under Different Induction Conditions.
[0172] To determine the best growth and protein induction conditions to use, pET29/TEV variants 9 and 10 (Table 3) were grown and induced in an IPTG induced media and an auto-inducing media. In addition, the length of induction was examined.
[0173] To induce expression with IPTG, cells harboring the TEV expression construct were first grown overnight to produce a starter culture. Fresh LB media was inoculated at 1:1000 with the overnight culture and allowed to grow, with shaking, at 37° C. until OD600 reached 0.7, at which time IPTG was added to a final concentration of 0.6 mM. Cells were harvested 4 hrs. following induction and total cell lysates evaluated to detect target expression.
[0174] To express constructs under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin was inoculated with a single colony of BL21(DE3) cells harboring the appropriate expression construct and grown at 37° C. with shaking overnight. 1.0 μL of this starter culture was used to inoculate 1.0 mL of ZYP-5052 auto-induction media containing 50 μg/mL kanamycin. Cells were grown at 37° C. with shaking and aliquots removed at 5, 8, 12, 20, and 28 hours.
[0175] To determine total TEV protease expression, 40 μL of the induced cell culture from each time-point was mixed with an equal volume of 2× Laemmi Sample Buffer and incubated at 95° C. for 10 minutes. 2 μL of 1 unit/μL Benzonase in 1 M MgSO4 was added to this mixture and incubated at 95° C. for 5 minutes. A 15 μL aliquot was loaded and separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions. The gel was washed and fixed in Fix Solution comprising 10% methanol, 7% acetic acid for 30 minutes. After fixing, the Fix Solution was removed and the gel incubated with SYPRO Ruby Protein Gel Stain at room temperature for 3 hours. The gel was then destained in Destain Solution comprising 10% methanol, 7% acetic acid at room temperature for 3 hours. The image was visualized with a Typhoon 9410 Variable Mode Imager and Imager Analysis software (GE Healthcare, Amersham Biosciences, Piscataway, N.J.).
[0176] To determine soluble TEV protease expression, 1.0 mL of the induced cell culture was lysed by adding 100 μL of a Cell Lysis Solution comprising 1× FASTBREAK® Cell Lysis reagent (Promega Corp., Madison, Wis.), 500 mM NaCl, 250 units/mL benzonase nuclease (EMD Biosciences-Novagen, Madison, Wis.), and 1× Protease Inhibitor Cocktail III (EMD Biosciences-Calbiochem, Gibbstown, N.J.) and incubated at room temperature for 25 minutes with constant vortexing. The lysate was centrifuged at 4300 rpm for 15 minutes to pellet debris. 800 μL of the supernatant was transferred to a clean tube, to which 30 μL of MagneHis magnetic beads were added and the mixture incubated for 5 minutes with constant rotation. After incubation, the magnetic beads were sequestered on a magnetic stand, the solution was removed, and the beads washed three times with 150 μL wash buffer comprising 500 mM NaCl. The protein was eluted with 80 μL of elution buffer, an equal volume of 2× Laemmli Sample Buffer was added, and the mixture incubated at 95° C. for 10 minutes. A 15 μL aliquot was loaded and separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions.
[0177] Results of the induction experiments indicated that auto-induction conditions resulted in 5-10-fold more expressed TEV protease relative to IPTG-induction. Comparison of total and soluble TEV protease expression in the auto-induction media revealed that although longer induction times resulted in more total protein, the amount of recoverable soluble TEV protease decreased. In fact, about 8 hours of expression at 37° C. yielded the largest amount of soluble protein. Lastly, although both the TEV S219N and TEV S219V variants exhibited significantly less autoproteolysis, the TEV S219V variant showed more truncated product at prolonged induction times suggesting that the TEV S219V variant was more prone to autoproteolysis.
[0178] Once the growth and induction conditions were optimized using pET29/TEV variants 9 and 10, expression of all eleven pET29/TEV variants was examined in parallel under these conditions. The results indicated that the order of increasing yield of soluble TEV protease, from greatest to least of the five highest expressers, was from pET29/TEV variants 5, 10, 7, 3, and 6. In comparison, the TEV variant 11 was expressed at the lowest level of all.
C. Large-Scale Expression and Purification.
[0179] To rigorously compare TEV protease expression levels from the top five pET29/TEV variants, along with variant 11 as a control, under large-scale conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL Kanamycin was inoculated with a single colony of BL21(DE3) cells harboring the appropriate expression construct and grown at 37° C. with shaking overnight. 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37° C. with shaking for 8 hours. The cells were pelleted by centrifugation.
[0180] To lyse cells, the cell pellet was resuspended in a 5.0 mL/gram cell pellet of Lysis Solution comprising BUGBUSTER® Protein Extraction Reagent (EMD Biosciences-Novagen, Madison, Wis.), 1× protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, Gibbstown, N.J.), 25 units/mL Benzonase nuclease, and 1 Kunit/mL rLysozyme (EMD Biosciences-Novagen, Madison, Wis.). The cell suspension was incubated at room temperature on a platform rocker for 20 minutes, followed by incubation on ice for 15 minutes. The suspension was centrifuged at 4° C. for 30 minutes at 30,350 rcf to pellet debris and the supernatant was transferred to a clean tube. To prepare the insoluble cell extract pellet for SDS-PAGE analysis, the pellet was resuspended to the original volume with 1× BUGBUSTER® Protein Extraction Reagent.
[0181] To purify a TEV protease variant by IMAC purification, the clarified lysate was mixed with TALON® SuperFlow Metal Affinity Cobalt Resin equilibrated with IMAC Wash Solution comprising 25 mM Sodium phosphate, pH 7.0, 500 mM NaCl, 10% glycerol and 35 mM imidazole. The lysate-resin mixture was incubated on a platform rocker at 4° C. for 1 hour and then transferred to a 20 mL disposable column support attached to a vacuum manifold. The column was washed twice with five column volumes of IMAC Wash Solution. The TEV protease was eluted from the resin with two column volumes of IMAC Elution Solution, comprising 25 mM sodium phosphate, pH 7.8, 500 mM NaCl, 10% glycerol and 500 mM imidazole, and collected in 1.0 mL fractions. Each fraction containing protein was identified by mixing 10 μL aliquot with 200 μL of QUICKSTART® Bradford Dye reagent. Peak elution fractions were pooled and dialyzed for secondary ion exchange chromatography purification.
[0182] To dialyze an IMAC-purified TEV protease variant, the pooled sample comprising the peak elution fraction was dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4° C. in 1 L of a Desalting Buffer with constant stirring overnight. For cation exchange chromatography, the desalting buffer (Buffer A) comprised 50 mM Tris-HCl, pH 8.0.
[0183] To purify a TEV protease variant by cation exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-S1 cation exchange column, pre-equilibrated with Buffer A, at a flow rate of 0.5 mL/min. Bound protein was eluted by NaCl gradient with Buffer B comprising 25 mM sodium phosphate, pH 7.0, 1 M NaCl at a flow rate of 1.0 mL/min as follows: 5% Buffer B for 3 mL, 20% Buffer B for 10 mL, 20% to 100% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were flash frozen in liquid nitrogen and stored at -80° C. TEV variant 7 had the highest yield of soluble protease (ca. 35 mg/L) followed by variant 3 (ca. 24 mg/L) and variant 10 (ca. 23 mg/L). The remaining two variants, 5 and 6, had yields of 18 and 8 mg/L, respectively. Yield of the TEV variant 11 was ca. 0.6 mg/L. As such, all of the top five TEV variants containing a solubility enhancing amino acid change resulted in at least a 10-fold increase in soluble TEV protease purified relative to the TEV variant 11 that only comprised the autoproteolysis eliminating amino acid change (S219N). When comparing the rank order of yield of TEV protease from small- and large-scale expression studies, variant 5 exhibited the highest yield in small-scale expressions (Example 1C). However, it was variant 7 that had the highest yield in large-scale expressions. Repeat comparison of yields from large-scale batches consistently revealed variant 7 to be the highest expressing variant. As a result, variant 7 represented the lead TEV protease construct and was used for all subsequent studies described here.
[0184] To determine the proteolytic activity of TEV protease variants, a TEV protease variant, or AcTEV protease as a positive control, was added to 30 μL of a Reaction Solution comprising 50 mM Tris-HCl, pH 8.0, 1 mM DTT, and 2.5 μg of a TEV substrate and incubated at 30° C. for 30 minutes, 60 minutes, and 120 minutes. The reactions were quenched by adding 2× Laemmi Sample Buffer and incubating the sample at 95° C. for 10 minutes. A 15 μL aliquot was loaded and separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions. The gel was washed and fixed in Fix Solution comprising 10% methanol, 7% acetic acid for 30 minutes. After fixing, the Fix Solution was removed and the gel incubated with SYPRO Ruby Protein Gel Stain at room temperature for 3 hours. The gel was then destained in Destain Solution comprising 10% methanol, 7% acetic acid at room temperature for 3 hours. The image was visualized with a Typhoon 9410 Variable Mode Imager and analyzed with ImageQuantTL Image Analysis software (GE Healthcare, Amersham Biosciences, Piscataway, N.J.). The ratio of intensities of uncleaved substrate and cleaved product was used to calculate percentage of cleaved TEV substrate. The results of the TEV protease activity assay are given in Table 4.
TABLE-US-00004 TABLE 4 TEV Protease Activity Assay TEV Substrate Cleavage (%) TEV Protease 30 minute 60 minute 120 minute AcTEV 73.9 91.6 97.2 TEV variant 3 96.5 97.7 98.1 TEV variant 5 95.6 97.8 95.6 TEV variant 6 90.8 96.8 97.2 TEV variant 7 96.6 97.8 97.7 TEV variant 10 74.2 93.3 96.1
Example 2
Intracellular Activation of a Clostridial Toxin with a TEV Protease Cleavage Site Using Two Different Expression Constructs
[0185] The following example illustrates a procedure useful for expressing in a cell a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification.
A. Construction of pET29/BoNT/A-TEV Expression Construct.
[0186] In order to produce a BoNT/A comprising a TEV protease cleavage site located within the di-chain loop region, an open reading frame (SEQ ID NO: 87) encoding the desired BoNT/A-TEV (SEQ ID NO: 88) was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, Wash.). Complementary oligonucleotides of 20 to 50 bases in length, spanning the entire open reading frame of BoNT/A-TEV were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHB1 carrier vector at the SmaI site to generate the pUCBHB1/BoNT/A-TEV constructs. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR® Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).
[0187] To generate the pET29/BoNT/A-TEV expression construct, pUCBHB1/BoNT/A-TEV was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding BoNT/A-TEV; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert was subcloned using a T4 DNA ligase procedure into a pET29 vector digested with the analogous restriction endonucleases to yield the appropriate pET29/BoNT/A-TEV expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding BoNT/A-TEV operably-linked to a carboxyl-terminal polyhistidine affinity purification peptide.
B. Construction of pET22/TEV Expression Constructs.
[0188] To generate a pET22/TEV variant expression construct, a pET29/TEV variant 7 expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 77) encoding the TEV protease (SEQ ID NO: 78); and 2) enable this insert to be operably-linked to a pET22 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert was subcloned using a T4 DNA ligase procedure into a pET22 vector digested with the analogous restriction endonucleases to yield the appropriate pET22/TEV expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pET22 expression construct comprising the polynucleotide molecule encoding TEV variant 7 operably-linked to an amino-terminal polyhistidine affinity purification peptide.
C. Construction of Cells Comprising pET29/BoNT/A-TEV and pET22/TEV Expression Constructs.
[0189] To make a cell comprising pET29/BoNT/A-TEV and pET22/TEV expression constructs, a pET29/BoNT/A-TEV expression construct was transformed into electro-competent E. coli BL21(DE3) cells harboring pET22/TEV variant 7 expression construct using electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin and 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing both expression constructs were identified as ampicillian-kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence of both constructs. This cloning strategy yielded cells comprising pET29/BoNT/A-TEV and pET22/TEV expression constructs.
D. In Situ Activation of BoNT/A-TEV.
[0190] To produce di-chain forms of BoNT/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin and 50 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells harboring pET29/BoNT/A-TEV and pET22/TEV expression constructs and grown at 37° C. with shaking overnight. About 1.0 μL of this starter culture was used to inoculate a 1.0 mL of ZYP-5052 containing 50 μg/mL kanamycin and 50 μg/mL ampicillin and grown at 37° C. with shaking for 3.5 hours and then at 22° C. with shaking for 18.5 hours. As a control, BL21(DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent.
[0191] Following growth and induction, the cells were lysed and IMAC purified essentially as described in Example 1B. The IMAC purified samples were analyzed by SDS-PAGE and the gels stained essentially as described in Example 1B.
[0192] The results indicate that when pET29/BoNT/A-TEV is expressed alone, an approximately 150 kDa band corresponding to the single-chain for of BoNT/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when BoNT/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50 kDa and the other of approximately 100 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50 kDa and approximately 100 kDa bands disappeared and a new band of approximately 150 kDa was observed. Taken together, these observations indicate that the approximately 50 kDa and approximately 100 kDa bands seen under reducing conditions correspond to the light and heavy chains of the BoNT/A-TEV, and that the presence of these two bands was indicative of di-chain formation of BoNT/A-TEV. Thus, co-expression of BoNT/A-TEV and TEV protease in these cells results in cleavage of BoNT/A-TEV at the TEV protease cleavage site located within the di-chain loop and the subsequent formation of the di-chain form of BoNT/A-TEV.
[0193] To confirm these results, a large scale expression of BL21(DE3) cells harboring pET29/BoNT/A-TEV and pET22/TEV expression constructs was done. 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin and 50 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells comprising pET29/BoNT/A-TEV and pET22/TEV expression constructs and grown at 37° C. with shaking overnight. About 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and 50 μg/mL ampicillin and grown at 37° C. with shaking for 3.5 hours and then at 22° C. with shaking for 18.5 hours. The cells were pelleted by centrifugation. The cells were lysed and IMAC purified as described in Example 1C.
[0194] To dialyze the IMAC-purified BoNT/A-TEV for secondary ion exchange chromatography, the pooled sample comprising the peak elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4° C. in 1 L of a Desalting Buffer with constant stirring overnight. For anion exchange chromatography, the desalting buffer (Buffer A) comprised 50 mM Tris-HCl, pH 8.0.
[0195] To purify BoNT/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 0.5 mL/min. Bound protein was eluted by NaCl gradient with Buffer B comprising 50 mM Tris-HCl, pH 8.0, 1 M NaCl at a flow rate of 0.5 mL/min as follows: 3% Buffer B for 3 mL, 7% Buffer B for 10 mL, 7% to 100% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were flash frozen in liquid nitrogen and stored at -80° C. Purified BoNT/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B. The results confirm the initial small scale experiments and indicate that the single-chain BoNT/A-TEV is converted to its di-chain form with near 100% efficiency.
[0196] To assess the activity of the BoNT/A-TEV di-chains, these toxins were evaluated in a cell-based assay and animal-based assay.
[0197] To test the activity of BoNT/A-TEV di-chains using a cell-based assay, an immuno-based BoNT/A activity assay using multiplex ECL sandwich ELISA was performed essentially as described in patent application Fernandez-Salas, et al., Immuno-Based BoNT/A Activity Assays, Attorney Docket No. 18383 (BOT), which is hereby incorporated by reference in its entirety.
[0198] To obtain a BoNT/A-TEV treated cell lysate for analysis, approximately 50,000 cells from a stock culture of a SiMa cell line were seeded into a poly-D-lysine 96-well plate containing a serum-free medium containing Minimum Essential Medium, 2 mM GlutaMAXT® I with Earle's salts, 1×B27 supplement, 1×N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mM HEPES and 25 μg/mL of GTb1. These cells were incubated in a 37° C. incubator under 5% carbon dioxide until the cells differentiated, as assessed by standard and routine morphological criteria, such as growth arrest and neurite extension (approximately 3 days). The media was aspirated from each well and replaced with fresh media containing either 0 (untreated sample), 0.01 nM, 0.04 nM, 0.12 nM, 0.37 nM, 1.11 nM, 3.33 nM and 10.0 nM of a BoNT/A-TEV. After a 24 hr treatment, the cells were washed, incubated for an additional two days without toxin. To harvest the cells, the medium was aspirated, washed with 1×PBS, and lysed by adding 30 μl of Lysis Buffer comprising 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100 to each well, and the plate incubated on a shaker rotating at 500 rpm for 30 minutes at 4° C. The plate was centrifuged at 4000 rpm for 20 minutes at 4° C. to pellet cell debris and the supernatant was transferred to a capture antibody coated 96-well plate to perform the detection step.
[0199] To prepare the α-SNAP-25 capture antibody solution, the α-SNAP-25 monoclonal antibody contained in the ascites from hybridoma cell line 2E2A6 was purified using a standard Protein A purification protocol To prepare the α-SNAP-25 detection antibody solution, α-SNAP-25 rabbit polyclonal antibody S9684 (Sigma, St. Louis, Mo.) was conjugated to Ruthenium(II)-tris-bipyridine-(4-methysulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, Md.) according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, Md.). To prepare the solid phase support comprising the capture antibody that was specific for a SNAP-25 cleaved product, approximately 5 μL of α-SNAP-25 monoclonal antibody 2E2A6 solution (20 μg/mL in 1×PBS) was added to each well of a 96-well MSD High Bind plate and the solution was allowed to air dry in a biological safety cabinet for 2-3 hours in order to liquid evaporate the solution. The capture antibody-bound wells were then blocked and used directly to detect BoNT/A activity.
[0200] To detect the presence of a cleaved SNAP-25 product by ECL sandwich ELISA analysis, the Blocking Buffer from stored plates was aspirated, 25 μL of a lysate from cells treated with BoNT/A was added to each well and the plates were incubated at 4° C. for 2 hrs. Plate wells were washed three times by aspirating the cell lysate and rinsing each well three times with 200 μL 1×PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). After washing, 25 μl of 5 μg/mL α-SNAP-25 detection antibody solution comprising 2% Amersham Blocking Reagent in 1×PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate) was added to each well, the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After α-SNAP-25 detection antibody incubation, the wells were washed three times with 200 μL 1×PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaureate). The raw data obtained from the ECL imager was then transferred to SigmaPlot v. 9.0 and a 4-parameter logistics fit was used to define the dose-response curves. There were no constraints used for the 4-parameter logistic function when plotting the data. Graphical reports were generated using the following analysis: R2 (correlation coefficient), a (Max for data set), b (hillslope), and X0±SE (EC50 value±standard error). The results from two independent runs indicate that the activity of both di-chains was nearly identical and within 2-fold of the native di-chain.
[0201] To test the activity of BoNT/A-TEV di-chains using an animal-based assay, an in vivo Digit Abduction Score (DAS) assay was performed. CD-1 Fe mice were weighed and placed into subsets of 10 animals for each discrete DAS assay. Mice were included into a particular subset based on the following criteria: 1) good health; 2) robust baseline DAS response of 0; 3) inclusion in a median weight range of X±2 g established for the selected subset and 4) weight greater than 17.0 g.
[0202] Each mouse was injected with 5 μL of one of seven different doses of BoNT/A-TEV (0.01 nM, 0.04 nM, 0.12 nM, 0.37 nM, 1.11 nM, 3.33 nM and 10.0 nM) with a 30-gauge needle in the gastrocnemius muscle of the right hind limb. As a control, the gastrocnemius muscle of the left hind limb was injected with 5 μL of a solution not containing any BoNT/A-TEV. Mice were observed for the DAS response consecutively for the first 4 days. The DAS was read by lifting each mouse by the tail and precisely observing the injected hind limbs. The abduction or no abduction of the hind digits reveals the effect of paralysis due to the test toxin injected in the muscle. The digit abduction of the injected hind limb was compared with that of the non-injected hind limb and scored accordingly. DAS data was analyzed by calculating the ED50 dose based on peak mean DAS score and AUC (area under the curve) in terms of u/Kg and/or ng/Kg. This was accomplished as follows: 1) the mean peak DAS score for each dose was calculated in each study; 2) any dose that elicited more than five deaths in any study was eliminated from consideration; 3) the highest dose used in a given individual study was the lowest dose which elicited an average peak of 4.0; 4) the lowest dose used in a given individual study was the highest dose which elicited an average peak of 0; 5) curves were constructed for each individual study of average peak DAS vs. log(dose); 6) an AUC value was calculated for each group of 10 mice of the multiple groups in some studies; 7) curves were constructed for each individual study of average AUC vs. log(dose); 8) an x, y replicate response curve was constructed for each set of multiple identical studies; for each test toxin; 9) dose-response data were analyzed by non-linear regression (non-weighted) using a three-parameter logistic equation (Sigma Plot v 8.0; SPSS Science, Chicago, Ill.) using the following equation:
y=a/(1+(x/x0)b)
where y is the response, a is the asymptotic ymax, b is the slope, x is the dose, and 0 is the ED50 dose, For peak ED50 determinations, Ymax was set to 4 (maximum DAS reading on scale). Mean (peak and/or AUC) ED50 values were computed for each eight-dose study performed.
[0203] The results from two independent runs indicate that the level of activity of both di-chains was nearly identical and within 2-fold of the native di-chain. Taken together, the cell-based assay and DAS assay data indicate that the process of intracellular activation yields di-chain rBoNT/A which was not only structurally comparable to the in-vitro nicked material but also functionally indistinguishable.
Example 3
Intracellular Activation of a Clostridial Toxin with a TEV Protease Cleavage Site Using Two Different Expression Constructs Under Control of Independent Promoters
[0204] The following example illustrates a procedure useful for expressing in a cell a Clostridial toxin comprising a di-chain loop region comprising an exogenous protease cleavage site as disclosed in the present specification. In this case, the formation of the di-chain form of the toxin is regulated by TEV protease under control of an independent promoter.
A. Construction of pBAD/TEV Expression Construct.
[0205] In order to produce a TEV protease recombinantly, the expression of which was under control of an arabinose promoter (PBAD), the open reading frame encoding the TEV protease variant 7 (Table 3 [130]), minus an N-terminal His tag, was cloned into the expression vector pBAD/Myc-HisA to construct pBAD/TEV. To construct pBAD/TEV, an open reading frame encoding the TEV protease variant 7 (SEQ ID NO: 106), minus an N-terminal poly-histidine tag, was synthesized using standard procedures (BlueHeron Biotechnology, Carlsbad, Calif.). The synthetic fragment was also flanked by restriction sites to enable this insert to be operably-linked to a pBAD/Myc-HisA vector (Life Technologies, Madison, Wis.). Using a T4 DNA ligase procedure this insert was directionally ligated into a pBAD/Myc-HisA vector digested with the same restriction endonucleases in the multiple cloning site. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the TEV gene insert. This cloning strategy yielded a pBAD/TEV expression construct comprising the polynucleotide molecule encoding TEV variant 7 free of a polyhistidine affinity purification peptide.
B. Construction of Cells Comprising pET29/BoNT/A-TEV and pBAD/TEV Expression Constructs.
[0206] To make a cell comprising pET29/BoNT/A-TEV and pBAD/TEV expression constructs, a pET29/BoNT/A-TEV expression construct (described in Example 2A) was transformed into electro-competent E. coli BL21(DE3) cells harboring pBAD/TEV variant 7 expression construct using electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin and 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing both expression constructs were identified as ampicillian-kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence of both constructs. This cloning strategy yielded cells comprising pET29/BoNT/A-TEV and pBAD/TEV expression constructs.
C. In Situ Activation of BoNT/A-TEV.
[0207] To produce di-chain forms of BoNT/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin and 50 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells harboring pET29/BoNT/A-TEV and pBAD/TEV expression constructs and grown at 37° C. with shaking overnight. 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and 100 μg/mL ampicillin and grown at 37° C. with shaking for 8 hours and then at 22° C. with shaking for 14 hours. At this point, TEV expression was induced with 0.2% L-arabinose and the culture was grown for an additional 4 hours at 22° C. As a control, BL21(DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent.
[0208] Following growth and induction, the cells were lysed and IMAC purified essentially as described in Example 1C. To dialyze the IMAC-purified BoNT/A-TEV for secondary ion exchange chromatography, the pooled sample comprising the peak elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4° C. in 1 L of a Desalting Buffer with constant stirring overnight. For anion exchange chromatography, the desalting buffer (Buffer A) comprised 50 mM Tris-HCl, pH 8.0.
[0209] To purify BoNT/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 0.5 mL/min. Bound protein was eluted by NaCl gradient with Buffer B comprising 50 mM Tris-HCl, pH 8.0, 1 M NaCl at a flow rate of 0.5 mL/min as follows: 3% Buffer B for 3 mL, 7% Buffer B for 10 mL, 7% to 100% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined.
[0210] Purified BoNT/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B. The results indicate that when pET29/BoNT/A-TEV is expressed alone, an approximately 150 kDa band corresponding to the single-chain for of BoNT/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when BoNT/A-TEV was co-expressed with TEV protease under control of the PBAD promoter and induced with arabinose, two bands were observed under reducing conditions, one of approximately 50 kDa and the other of approximately 100 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50 kDa and approximately 100 kDa bands disappeared and a new band of approximately 150 kDa was observed. Taken together, these observations indicate that the approximately 50 kDa and approximately 100 kDa bands seen under reducing conditions correspond to the light and heavy chains of the BoNT/A-TEV, and that the presence of these two bands was indicative of di-chain formation of BoNT/A-TEV. Thus, co-expression of BoNT/A-TEV and TEV protease in these cells results in cleavage of BoNT/A-TEV at the TEV protease cleavage site located within the di-chain loop and the subsequent formation of the di-chain form of BoNT/A-TEV. The results indicate that between 90-95% of the single-chain BoNT/A-TEV is converted to its di-chain form.
Example 4
Intracellular Activation of a Clostridial Toxin with a TEV Protease Cleavage Site using a Dual Expression Construct
[0211] The following example illustrates methods useful for purifying and quantifying a Clostridial toxin comprising an exogenous protease cleavage site as disclosed in the present specification.
A. Construction of pET29/BoNT/A-TEV/2×TEV Dual Expression Construct.
[0212] To construct pET29/BoNT/A-TEV/2×TEV dual expression construct, a synthetic fragment (SEQ ID NO: 89) encoding the last 37 amino acids of BoNT/A-TEV as well as transcription (T7 promoter, lac operator site) and translation (RBS) elements necessary for E. coli expression and the entire coding region of TEV variant 7 was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, Wash.). Complementary oligonucleotides of 20 to 50 bases in length, were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHB1 carrier vector at the SmaI site to generate the pUCBHB1/BoNT/A-TEV_C-term/T7Prom/TEV plasmid. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR® Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.
[0213] To generate the pET29/BoNT/A-TEV/2×TEV expression construct, pUCBHB1/BoNT/A-TEV_C-term/T7Prom/TEV was digested with restriction endonucleases that 1) excise the insert comprising the C-terminus of BoNT/A-TEV, transcription and translation motifs necessary for E. coli expression of a second open reading frame, and the entire coding region of TEV variant 7; and 2) enable this insert to be operably-linked behind the BoNT/A gene in pET29/BoNT/A-TEV vector from Example 1A. This insert was subcloned using a T4 DNA ligase procedure into the pET29/BoNT/A-TEV vector digested with the analogous restriction endonucleases to yield the appropriate pET29/BoNT/A-TEV/2×TEV dual expression construct comprising the BoNT/A-TEV and TEV protease variant 7 open reading frames with the intervening transcription and translation elements of SEQ ID NO: 89. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pET29 dual expression construct comprising the polynucleotide molecule encoding a BoNT/A-TEV variant operably-linked to a carboxyl terminal polyhistidine affinity purification tag and a TEV protease. The open reading frame organization was such that transcription initiation from the first T7 promoter yields an mRNA with the open reading frame encoding BoNT/A-TEV and the open reading frame encoding TEV protease. In addition, transcription initiation from the second T7 promoter yields mRNA with the open reading frame encoding only TEV protease. Thus, there would be twice as many transcripts encoding TEV protease compared to BoNT/A-TEV.
B. In Situ Activation of BoNT/A-TEV from pET29/BoNT/A-TEV/2×TEV.
[0214] To produce di-chain forms of BoNT/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL Kanamycin was inoculated with a single colony of BL21(DE3) cells comprising pET29/BoNT/A-TEV/TEV dual expression construct and grown at 37° C. with shaking overnight. About 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37° C. with shaking for 3.5 hours and then at 22° C. with shaking for 18.5 hours. The cells were pelleted by centrifugation. The cells were lysed, IMAC purified, desalted, purified by anion exchange chromatography, analyzed by SDS-PAGE, and the gels stained essentially as described in Example 2D. As a control, BL21(DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent.
[0215] The results indicate that when expressed alone, an approximately 150 kDa band corresponding to the single-chain for of BoNT/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when BoNT/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50 kDa and the other of approximately 100 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50 kDa and approximately 100 kDa bands disappeared and a new band of approximately 150 kDa was observed. Taken together, these observations indicate that the approximately 50 kDa and approximately 100 kDa bands seen under reducing conditions correspond to the light and heavy chains of the BoNT/A-TEV, and that the presence of these two bands was indicative of di-chain formation of BoNT/A-TEV. The results also indicated that the single-chain BoNT/A-TEV was converted to its di-chain form with greater than 95% efficiency. Thus, co-expression of BoNT/A-TEV and TEV protease from a dual expression construct in these cells results in cleavage of BoNT/A-TEV at the TEV protease cleavage site located within the di-chain loop and the subsequent formation of the di-chain form of BoNT/A-TEV.
C. Construction of pRSFduet/TEV/2×BoNT/A-TEV Dual Expression Constructs.
[0216] To determine if reversing the organization of the open reading frames encoding BoNT/A-TEV and the TEV protease would affect yield and cleavage efficiency of BoNT/A-TEV, a dual expression construct was made where transcription initiation from the first T7 promoter yields an mRNA with the open reading frames encoding TEV and BoNT/A-TEV and transcription initiation from the second T7 promoter yields mRNA with the open reading frame encoding only BoNT/A-TEV. Thus, there would be twice as many mRNA's encoding BoNT/A-TEV compared to TEV protease.
[0217] To construct pRSFduetJTEV/2×BoNT/A-TEV dual expression construct, two sequential cloning reactions were performed. First, the open reading frame (SEQ ID NO: 91) encoding TEV variant 7 (SEQ ID NO: 22) was amplified by PCR from the pET29/TEV variant 7 expression construct. The 5'-end of the open reading frame encoding the poly-histidine affinity tag was excluded from the amplification to encode a tag-less protease. Following amplification, the PCR product was digested at the unique restriction sites, incorporated at the ends of the PCR product by means of the PCR primers, and cloned into the corresponding sites in MCSI (multiple cloning site) of the dual expression plasmid pRSFduet-1 (EMD Biosciences-Novagen, Madison, Wis.) using a T4 DNA ligase procedure. This intermediate construct was designated pRSduetJTEV. Next, a pET29/BoNT-A/TEV expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 87) encoding the BoNT/A-TEV (SEQ ID NO: 88); and 2) enable this insert to be operably-linked to the MCS2 in pRSFduet/TEV. The BoNT/A-TEV insert was subcloned into the MCS2 of the pRSFduet vector using a T4 DNA ligase procedure to yield the appropriate pRSFduetJTEV/2×BoNT/A-TEV dual expression construct. This cloning strategy yielded a pRSFduet dual expression construct where transcription from the first T7 promoter would produce mRNA's encoding TEV and BoNT/A-TEV and transcription from the second T7 promoter would produce mRNA's encoding only BoNT/A-TEV.
[0218] This cloning strategy will yield a pRSFduet dual expression construct where the first T7 promoter will transcribe the open reading frame encoding BoNT/A-TEV and the second T7 promoter will transcribe the open reading encoding TEV protease.
D. Construction of pET29/BoNT/A-TEVITEV Dual Expression Construct.
[0219] To determine BoNT/A-TEV yields and efficiency of conversion to di-chain from a transcription unit configuration where BoNT/A-TEV and TEV could only be produced from their own independent mRNA's, pET29/BoNT/A-TEV/TEV was constructed. To generate the pET29/BoNT/A-TEV/TEV dual expression construct, a short synthetic DNA fragment was used to incorporate a T7 terminator site (SEQ ID NO: 92) in the intervening sequence between the open reading frames of BoNT/A-TEV and TEV in the dual expression construct pET29/BoNT/A-TEV/2×TEV (Example 3A above). Using a T4 DNA ligase procedure, this was essentially accomplished by swapping the intervening region in pET29/BoNT/A-TEV/2×TEV which lacked a T7 terminator site with a synthetic DNA fragment harboring the intervening transcription and translation elements along with a T7 termination site of SEQ ID NO: 93. The resulting dual expression construct, designated pET29/BoNT/A-TEV/TEV, comprises the polynucleotide molecule encoding a BoNT/A-TEV variant operably-linked to a carboxyl terminal polyhistidine affinity tag and TEV protease, transcribed from the first and second T7 promoters, respectively.
E. In Situ Activation of BoNT/A-TEV.
[0220] The growth and induction of di-chain forms of BoNT/A-TEV under auto-induction conditions was done essentially as described in Example 2D, except the BL21(DE3) cells comprising a pET29/BoNT/A-TEV/2×TEV dual expression construct, a pRSF/TEV/2×BoNT/A-TEV dual expression construct, or a pET29/BoNT/A-TEV/TEV dual expression construct were used and single colonies from each of these cell lines were used to inoculate four 1.0 mL cultures in parallel. After growth and induction, the four 1.0 mL replicates were pooled together for processing. The cells were lysed and IMAC purified, and analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B. As a control, BL21(DE3) cells harboring pET29/BoNT/A-TEV alone were grown and induced as described above, except only 50 μg/mL kanamycin was used as a selective agent. The results indicate that BoNT/A-TEV was expressed at very comparable levels from cells containing any one of the three dual expression constructs; however, the extent of conversion to di-chain varied. Single-chain BoNT/A-TEV was converted to its di-chain form with ca. 96% efficiency when the proteins were expressed from pET29/BoNT/A-TEV/2×TEV, with ca. 81% efficiency when the proteins were expressed from pET29/BoNT/A-TEV/TEV, and with greater than 99% efficiency when the proteins were expressed from pRSFduetJTEV/2×BoNT/A-TEV.
Example 5
Intracellular Activation of a Protein Comprising an Integrated TEV Protease Cleavage Site-Opioid Binding Domain Using a Dual Expression Construct
[0221] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an exogenous protease cleavage site disclosed in the present specification.
A. Construction of pRSFduet/TEV/2×NociLHN/A-TEV Dual Expression Construct.
[0222] To construct pRSFduet/TEV/2×NociLHN/A-TEV dual expression construct, a pET29/NociLHN/A-TEV expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 94) encoding the NociLHN/A-TEV (SEQ ID NO: 95); and 2) enable this insert to be operably-linked to the MCS2 of pRSFduet/TEV, a pRSFduet-1 vector harboring TEV variant 7 in MCSI (Described in Example 3C). The NociLHN/A-TEV insert was subcloned into the MCS2 of the pRSFduet/TEV construct using a T4 DNA ligase procedure to yield the appropriate pRSFduetJTEV/2×NociLHN/A-TEV dual expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pRSFduet dual expression construct where transcription from the first T7 promoter would produce mRNA's encoding TEV and NociLHN/A-TEV and transcription from the second T7 promoter would produce mRNA's encoding only NociLHN/A-TEV.
B. In Situ Activation of NociLHN/A-TEV.
[0223] To produce di-chain forms of NociLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin was inoculated with a single colony of BL21(DE3) cells comprising pRSFduetJTEV/2×NociLHN/A-TEV dual expression construct and grown at 37° C. with shaking overnight. 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37° C. with shaking for 8 hours and then at 16° C. with shaking for 18 hours. The cells were pelleted by centrifugation. The cells were lysed, IMAC purified, desalted, purified by anion exchange chromatography, analyzed by SDS-PAGE, and the gels stained essentially as described in Example 2D. As a control, BL21(DE3) cells harboring NociLHN/A-TEV alone were grown and induced as described above.
[0224] The results indicate that when expressed alone, an approximately 102 kDa band corresponding to the single-chain of NociLHN/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when NociLHN/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50.8 kDa and the other of approximately 51.3 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50.8 kDa and approximately 51.3 kDa bands disappeared and a new band of approximately 102 kDa was observed. Taken together, these observations indicate that the approximately 50.8 kDa and approximately 51.3 kDa bands seen under reducing conditions respectively correspond to the Clostridial toxin enzymatic domain and the Clostridial toxin translocation domain with the nociceptin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of NociLHN/A-TEV and that the single-chain NociLHN/A-TEV was converted to its di-chain form with greater than 95% efficiency. Thus, co-expression of NociLHN/A-TEV and TEV protease from a dual expression construct in these cells results in cleavage of NociLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-opioid binding domain and the subsequent formation of the di-chain form of NociLHN/A-TEV.
C. Construction of pRSFduet/TEV/2×DynLHN/A-TEV Dual Expression Construct.
[0225] pRSFduet/TEV/2×DynLHN/A-TEV dual expression construct was generated almost exactly as pRSFduetJTEV/2×NociLHN/A-TEV. A pET29/DynLHN/A-TEV expression construct was digested with restriction endonucleases that 1) excise the insert comprising the open reading frame (SEQ ID NO: 96) encoding the DynLHN/A-TEV (SEQ ID NO: 97); and 2) enable this insert to be operably-linked to the MCS2 of pRSFduet/TEV (Described in Example 3C). The DynLHN/A-TEV insert was subcloned into the MCS2 of the pRSFduet/TEV construct using a T4 DNA ligase procedure to yield the appropriate pRSFduetJTEV/2×DynLHN/A-TEV dual expression construct. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pRSFduet dual expression construct where transcription from the first T7 promoter would produce mRNA's encoding TEV and DynLHN/A-TEV and transcription from the second T7 promoter would produce mRNA's encoding only DynLHN/A-TEV.
D. In Situ Activation of DynLHN/A-TEV.
[0226] To produce di-chain forms of NociLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin was inoculated with a single colony of BL21(DE3) cells comprising pRSFduet/TEV/2×DynLHN/A-TEV dual expression construct and grown at 37° C. with shaking overnight. 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37° C. with shaking for 8 hours and then at 16° C. with shaking for 18 hours. The cells were pelleted by centrifugation. The cells were lysed, IMAC purified, desalted, purified by anion exchange chromatography, analyzed by SDS-PAGE, and the gels stained essentially as described in Example 2D. As a control, BL21(DE3) cells harboring DynLHN/A-TEV alone were grown and induced as described above.
[0227] The results indicate that when expressed alone, an approximately 102 kDa band corresponding to the single-chain for of DynLHN/A-TEV was detected under both reducing and non-reducing conditions. In contrast, when DynLHN/A-TEV was co-expressed with TEV protease, two bands were observed under reducing conditions, one of approximately 50.8 kDa and the other of approximately 52 kDa. Moreover, when the same samples were run under non-reducing conditions, the approximately 50.8 kDa and approximately 52 kDa bands disappeared and a new band of approximately 102 kDa was observed. Taken together, these observations indicate that the approximately 50.8 kDa band corresponds to the Clostridial toxin enzymatic domain and an approximately 52 kDa band corresponds to the Clostridial toxin translocation domain with the dynorphin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of DynLHN/A-TEV and also that the single-chain DynLHN/A-TEV was converted to its di-chain form with greater than 95% efficiency. Thus, co-expression of DynLHN/A-TEV and TEV protease from a dual expression construct in these cells results in cleavage of DynLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-opioid binding domain and the subsequent formation of the di-chain form of DynLHN/A-TEV.
Example 6
Intracellular Activation of a Protein Comprising an Integrated TEV Protease Cleavage Site-Galanin Binding Domain Using Two Different Expression Constructs
[0228] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an integrated TEV protease cleavage site-opioid binding domain disclosed in the present specification where the target protein and the protease are expressed from separate plasmids and under control of different promoters.
A. Construction of pET29/GalLHN/A-TEV Expression Construct.
[0229] To construct the pET29/GalLHN/A-TEV expression construct, a pET29/DynLHN/A-TEV expression construct was first digested with restriction endonucleases to excise a DNA segment encoding the di-chain loop comprising an integrated TEV protease cleavage site-dynorphin binding domain. The resulting pET29/LHn/A framework fragment was ligated with a synthetic DNA fragment bracketed with the compatible restriction sites (SEQ ID NO: 98), comprising the di-chain loop comprising an integrated TEV protease cleavage site-galanin binding domain (SEQ ID NO: 99). The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded the pET29/GalLHN/A-TEV expression construct comprising the open reading frame (SEQ ID NO: 100) encoding the GalLHN/A-TEV (SEQ ID NO: 101) in which expression of GalLHN/A-TEV is under control of the T7 promoter.
B. Construction of pColdIV/TEV Expression Construct.
[0230] To generate an expression construct in which TEV is under control of the cold-shock promoter (csp), the open reading frame (SEQ ID NO: 91) encoding TEV variant 7 (SEQ ID NO: 22) was amplified by PCR from the pET29/TEV variant 7 expression construct. The 5'-end of the open reading frame encoding the poly-histidine affinity tag was excluded from the amplification to encode a tag-less protease. Following amplification, the PCR product was digested at the unique restriction sites, incorporated at the ends of the PCR product by means of the PCR primers, and cloned into the corresponding sites in the multiple cloning site of the expression plasmid pColdIV (Clontech Laboratories, Inc. Madison, Wis.) using a T4 DNA ligase procedure. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of ampicillin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded the pColdIV/TEV expression construct comprising the polynucleotide molecule encoding TEV variant 7 under control of the cold-shock promoter.
C. Construction of Cells Comprising pET29/GalLHN/A-TEV and pColdIV/TEV Expression Constructs.
[0231] To make a cell comprising pET29/GalLHN/A-TEV and pColdIV/TEV expression constructs, the pET29/GalLHN/A-TEV expression construct was transformed into electro-competent E. coli BL21(DE3) cells harboring pColdIV/TEV using electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of ampicillin and 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing both expression constructs were identified as ampicillian-kanamycin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence of both constructs. This cloning strategy yielded cells comprising pET29/GalLHN/A-TEV and pColdIV/TEV expression constructs.
D. In Situ Activation of pET29/GalLHN/A.
[0232] To produce di-chain forms of GalLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin and 100 μg/mL ampicillin was inoculated with a single colony of BL21(DE3) cells harboring pET29/GalLHN/A-TEV and pColdIV/TEV expression constructs and grown at 37° C. with shaking overnight. About 250 μL of this starter culture was used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and 100 μg/mL ampicillin and grown at 37° C. with shaking for 8 hours and then at 15° C. with shaking for 18 hours. The cells were lysed and IMAC purified using Magne-His resin.
[0233] To purify di-chain GalLHN/A-TEV by Magne-His purification, induced cells from 250 mL expression cultures were resuspended in 16 mL of cold (4-6° C.) IMAC Wash Buffer consisting of 100 mM HEPES, pH 7.5, 10% v/v glycerol, 10 mM imidazole, 1 M NaCl. The cell suspension was transferred to a sealed-atmosphere treatment chamber (#101-021-006, Branson Ultrasonics Corporation) and sonicated by 15 pulses (10 sec, 30% amplitude, 0.5-inch disruptor horn) with 1 minute in between pulses (Sonifier® Digital 450, Branson Ultrasonics Corporation). During sonication the sealed-atmosphere treatment chamber was cooled by passing chilled water from a circulating water bath (3.5° C.) through the outer jacket of the chamber. Sonicated material was transferred from the treatment chamber to a clean Oakridge tube and centrifuged at 30,500 RCF for 30 min (SL-50T Rotor, Sorvall; FIBERLite® F21S-8×50 Rotor, Piramoon Technologies Inc.) at 4° C. to remove insoluble cellular debris. The clarified lysate was aspirated by syringe and passed first through a 0.8 μm and then a 0.45 μm syringe filter (Sartorius) in series into a clean 50 mL conical tube. Magne-His® Protein Purification Resin (Promega Corp., Madison, Wis.) was vortexed to a uniform suspension and 4 mL of the suspension transferred to the clarified lysate. The tube was sealed and inverted several times to mix the particles well. The mixture was incubated for 30 min with gentle rocking to bind the target protein at 16° C. The tube was transferred to a MagneSil Magnetic Separation Unit (Promega Corp., Madison, Wis.) and ˜2 min were allowed for capture of the resin particles. The supernatant solution was removed and the tube removed from the separation unit. The resin was then resuspended in 10 mL IMAC Wash Buffer, captured on the magnetic separation unit, and the wash buffer removed. The wash step was repeated two more times. To elute the target protein, the resin was resuspended in 5 mL of the Magne-His® Elution Buffer (100 mM HEPES, pH 7.5, 500 mM Imidazole) incubated at room temperature for 2 min, the resin captured on the magnetic separation unit and the supernatant solution transferred to a new tube. The elution step was repeated once.
[0234] To dialyze the IMAC-purified GalLHN/A-TEV for secondary ion exchange chromatography, the pooled elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4° C. in 1 L of a Desalting Buffer (Buffer A: 50 mM Tris-HCl, pH 8.0) with constant stirring overnight.
[0235] To purify di-chain GalLHN/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 1 mL/min. Bound protein was eluted by NaCl gradient with Buffer B comprising 50 mM Tris-HCl, pH 8.0, 1 M NaCl at a flow rate of 1 mL/min as follows: 7% Buffer B for 3 mL, 15% Buffer B for 7 mL, 10% to 50% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were flash frozen in liquid nitrogen and stored at -80° C. Purified BoNT/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B.
[0236] The results indicate that when GalLHN/A-TEV was co-expressed with TEV protease, two nearly superimposing bands were observed under reducing conditions, one of approximately 51.1 kDa and another of approximately 52.1 kDa. Moreover, when the same samples were run under non-reducing conditions, the two approximately 51.1 kDa and 52.1 kDa bands disappeared and a new band of approximately 103 kDa was observed. Taken together, these observations indicate that the approximately 51.1 kDa band corresponds to the Clostridial toxin enzymatic domain and the approximately 52.1 kDa band corresponds to the Clostridial toxin translocation domain with the galanin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of GalLHN/A-TEV and also that the single-chain GalLHN/A-TEV was converted to its di-chain form with approximately 90% efficiency. Thus, co-expression of GalLHN/A-TEV and TEV protease in these cells from independent plasmids results in cleavage of GalLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-galanin binding domain and the subsequent formation of the di-chain form of GalLHN/A-TEV.
Example 7
Prophetic Intracellular Activation of a Protein Comprising an Integrated TEV Protease Cleavage Site-Galanin Binding Domain Using a Dual Expression Construct
[0237] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an integrated TEV protease cleavage site-opioid binding domain disclosed in the present specification where the target protein and the protease are expressed from a dual expression plasmid.
A. Construction of pRSFduet/TEV/2×GalLHN/A-TEV Dual Expression construct.
[0238] To construct pRSFduet/TEV/2×GalLHN/A-TEV dual expression construct similar to pRSFduet/TEV/2×NociLHN/A-TEV and pRSFduet/TEV/2×DynLHN/A-TEV constructed before (See Example 4), a pET29/GalLHN/A-TEV expression construct will be digested with restriction endonucleases to 1) excise the insert comprising the open reading frame (SEQ ID NO: 100) encoding the GalLHN/A-TEV (SEQ ID NO: 101); and 2) enable this insert to be operably-linked to the MCS2 of pRSFduet/TEV, a pRSFduet-1 vector harboring TEV variant 7 in MCSI (Described in Example 3C). The GalLHN/A-TEV insert will be subcloned into the MCS2 of the pRSFduet/TEV construct using a T4 DNA ligase procedure to yield the pRSFduet/TEV/2×GalLHN/A-TEV dual expression construct. The ligation mixture will be transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs will be identified as kanamycin resistant colonies and candidate constructs confirmed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy will yield a pRSFduet dual expression construct where transcription from the first T7 promoter will produce mRNA's encoding TEV and GalLHN/A-TEV and transcription from the second T7 promoter will produce mRNA's encoding only GalLHN/A-TEV.
B. In Situ Activation of GalLHN/A-TEV.
[0239] To produce di-chain forms of GalLHN/A-TEV under auto-induction conditions, 3.0 mL of PA-0.5G media containing 50 μg/mL kanamycin will be inoculated with a single colony of BL21(DE3) cells comprising pRSFduet/TEV/2×GalLHN/A-TEV dual expression construct and grown at 37° C. with shaking overnight. 250 μL of this starter culture will be used to inoculate 250 mL of ZYP-5052 containing 50 μg/mL kanamycin and grown at 37° C. with shaking for 8 hours and then at 16° C. with shaking for 18 hours. The cells will be pelleted by centrifugation, lysed, IMAC purified, desalted, and purified by anion exchange chromatography as described in Example 5D. Purified target protein will be analyzed by SDS-PAGE under both reducing and non-reducing conditions, and the gels stained essentially as described in Example 1B to assess expression levels and the extent to which GalLHN/A-TEV produced from the pRSFduet/TEV/2×GalLHN/A-TEV dual expression construct is converted to its di-chain form.
Example 8
Intracellular Activation of a Protein Comprising an Integrated TEV Protease Cleavage Site-Dynorphin Binding Domain Using a Dual Expression Construct in BEVS
[0240] The following example illustrates methods useful for purifying and quantifying any of the proteins comprising a di-chain loop comprising an integrated TEV protease cleavage site-opioid binding domain disclosed in the present specification where the target protein and the protease are co-expressed in a dual expression construct and under control of two independent promoters in the baculovirus expression vector system (BEVS).
A. Construction of pBAC-6/TEV/DynLHN/A-TEV Dual Expression Construct.
[0241] To construct the pBAC-6/TEV/DynLHN/A-TEV dual expression construct, a synthetic fragment (SEQ ID NO: 107) encoding recombinant TEV variant 7 downstream of the p10 promoter sequence and DynLHn/A-TEV downstream of the polH promoter sequence in the opposite orientation was synthesized using standard procedures (BlueHeron Biotechnology, Bothell, Wash.). Complementary oligonucleotides of 20 to 50 bases in length were synthesized using standard phosphoramidite synthesis. These oligonucleotides were hybridized into double stranded duplexes that were sequentially ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule was cloned using standard molecular biology methods into a pUCBHB1 carrier vector at the SmaI site to generate the pUCBHB1/p10-TEV/polH-DynLHN/A-TEV plasmid. The synthesized polynucleotide molecule was verified by sequencing using BIG DYE TERMINATOR® Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.
[0242] To generate the pBAC-6/TEV/DynLHN/A-TEV dual expression construct, pUCBHB1/p10-TEV/polH-DynLHN/A-TEV was digested with restriction endonucleases that 1) excise the insert comprising the entire coding region of TEV variant 7 under control of the p10 promoter and DynLHN/A-TEV in the opposite direction under control of the polH promoter; and 2) enable this insert to be operably-linked to a pBAC-6 transfer vector (EMD Biosciences-Novagen, Madison, Wis.). This insert was subcloned using a T4 DNA ligase procedure into the pBAC-6 transfer vector digested with the analogous restriction endonucleases to yield the engineered pBAC-6 dual expression construct comprising TEV protease variant 7 open reading frame downstream of the p10 promoter and a second open reading frame of DynLHN/A-TEV downstream of the polH promoter. The ligation mixture was transformed into electro-competent E. coli BL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) by electroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of ampicillin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression construct were identified as ampicillin resistant colonies. Candidate constructs were isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping and sequencing both DNA strands to confirm the presence and integrity of the insert. This cloning strategy yielded a pBAC-6 dual expression construct comprising the polynucleotide molecule encoding a DynLH/A-TEV operably-linked to a carboxyl terminal polyhistidine affinity purification tag and TEV protease.
B. Generation of High Titer TEV/DynLHN/A-TEV Recombinant Baculovirus Stock.
[0243] Before di-chain forms of DynLHN/A-TEV could be produced, high titre recombinant baculovirus stock comprising TEV/DynLHN/A-TEV were generated. Approximately 2×106 Sf9 insect cells were seeded in 35 mm dishes in a 2 mL volume of insect cell culture medium ESF921. A transfection solution was prepared by mixing Solution A (comprising 2 μg of pBAC-6/TEV/DynLHN/A-TEV, 0.5 μg of linearized flashBAC baculovirus DNA (Oxford Expression Technologies, Oxford, UK), and 100 μL of Transfection Medium) with solution B (comprising 6 μL of TRANSLT®-2020 transfection reagent and 100 μL of Transfection Medium) and incubating at room temperature for 30 minutes. An additional 800 μL of Transfection Medium was next added to the Solution A/B mixture, mixed gently, and added dropwise onto the cells. Cells were incubated at 28° C. for 5 hours, at the end which 3 mL of ESF 921 was added to bring the final volume up to 4 mL in each well. The incubation was continued at 28° C. for 4-5 days for the production of P0 recombinant virus. To generate higher titer P1 recombinant baculovirus seed stocks, virus isolated from P0 supernatant was titered using bacu/oQUANT (Oxford Expression Technologies, Oxford, UK) and further amplified in shake flasks. About 100-200 mL of Sf9 cells at a density of 2×106 cells/mL were infected with P0 virus at an MOI (multiplicity of infection)<1 pfu/cell and incubated with shaking for 4-5 days. Following quantification, the high titer P1 stock was used to infect Tni cells for high-level protein expression.
C. In Situ Activation of DynLHN/A-TEV.
[0244] To produce di-chain forms of DynHN/A-TEV, 50 mL Tni cells at a concentration of 1×106/mL were infected at an MOI of 5 with recombinant P1 virus stock comprising TEV/DynLHN/A-TEV and harvested 3 days post-infection (pi). The cells were lysed and IMAC purified using Magne-His resin.
[0245] To purify di-chain DynLHN/A-TEV by Magne-His purification, the cell pellet was resuspended in 20 mL of PBS w/o Ca2+ or Mg2+ in the presence of 100 μL Insect PopCulture Reagent and 20 μL (10U) Benzonase Nuclease, mixed gently and incubated for 15 minutes at room temperature. After clarifying the cell lysate by centrifugation at 16,000 rpm for 15 minutes at 4° C., the supernatant was mixed with 4 mL of uniformly suspended Magne-His® Protein Purification Resin (Promega Corp., Madison, Wis.). The mixture was incubated for 20 min at room temperature with gentle rocking to bind the target protein. The tube was transferred to a MagneSil magnetic separation unit for about 2 min to allow capture of the resin particles. After removing the supernatant, the tube was removed from the separation unit and the resin resuspended in 10 mL of IMAC wash buffer. Again, the resin was captured on the magnetic separation unit and the wash buffer removed. The wash step was repeated two more times. To elute the target protein, the resin was resuspended in 2.5 mL of the Magne-His® Elution Buffer (100 mM HEPES, pH 7.5, 500 mM Imidazole), incubated at room temperature for 2 min, the resin captured on the magnetic separation unit, and the supernatant solution transferred to a new tube. The elution step was repeated again to maximize target recovery from the magnetic resin.
[0246] To dialyze the IMAC-purified DynLHN/A-TEV for secondary ion exchange chromatography, the pooled elution fractions were dialyzed in a FASTDIALYZER® fitted with 25 kD MWCO membrane at 4° C. in 1 L of a Desalting Buffer (Buffer A: 50 mM Tris-HCl, pH 8.0) with constant stirring overnight.
[0247] To purify di-chain DynLHN/A-TEV by anion exchange chromatography, the desalted protein solution was loaded onto a 1 mL UNO-Q1 anion exchange column, pre-equilibrated with Buffer A, at a flow rate of 1 mL/min. Bound protein was eluted by NaCl gradient with Buffer B comprising 50 mM Tris-HCl, pH 8.0, 1 M NaCl at a flow rate of 1 mL/min as follows: 7% Buffer B for 3 mL, 15% Buffer B for 7 mL, 10% to 50% Buffer B over 10 mL. Elution of proteins from the column was detected with a UV-Visible detector at 214 nm, 260 nm, and 280 nm, and all peak fractions were pooled and protein concentration determined. Aliquots were stored at -20° C. Purified DynLHN/A-TEV protein was analyzed by SDS-PAGE, and the gels stained essentially as described in Example 1B.
[0248] The results indicate that when DynLHN/A-TEV was co-expressed with TEV protease in insect cells and purified to near homogeneity, two nearly superimposing bands were observed under reducing conditions, one of approximately 51 kDa and another of approximately 52 kDa. Moreover, when the same samples were run under non-reducing conditions, the two approximately 50 kDa and 52 kDa bands disappeared and a new band of approximately 102 kDa was observed. Taken together, these observations indicate that the approximately 51 kDa band corresponds to the Clostridial toxin enzymatic domain and the approximately 52 kDa band corresponds to the Clostridial toxin translocation domain with the dynorphin targeting moiety attached to its amino terminus. The presence of these two bands was indicative of di-chain formation of DynLHN/A-TEV and also that the single-chain DynLHN/A-TEV was converted to its di-chain form with 80-90% efficiency. Thus, co-expression of DynLHN/A-TEV and TEV protease in insect cells infected with TEV/DynLHN/A-TEV recombinant baculovirus generated from pBAC-6/TEV/DynLHN/A-TEV dual expression construct results in cleavage of DynLHN/A-TEV at the TEV protease cleavage site located within the integrated TEV protease cleavage site-dynorphin binding domain and the subsequent formation of the di-chain form of DynLHN/A-TEV.
[0249] Although aspects of the present invention have been described with reference to the disclosed embodiments, one skilled in the art will readily appreciate that the specific examples disclosed are only illustrative of these aspects and in no way limit the present invention. Various modifications can be made without departing from the spirit of the present invention.
Sequence CWU
1
1
10711296PRTClostridium botulinum Serotype A 1Met Pro Phe Val Asn Lys Gln
Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10
15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly
Gln Met Gln Pro 20 25 30
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg
35 40 45 Asp Thr Phe Thr
Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55
60 Ala Lys Gln Val Pro Val Ser Tyr Tyr
Asp Ser Thr Tyr Leu Ser Thr65 70 75
80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu
Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val
100 105 110 Arg Gly Ile Pro Phe
Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115
120 125 Val Ile Asp Thr Asn Cys Ile Asn Val
Ile Gln Pro Asp Gly Ser Tyr 130 135
140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser
Ala Asp Ile145 150 155
160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr
165 170 175 Arg Asn Gly Tyr
Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180
185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu
Val Asp Thr Asn Pro Leu Leu 195 200
205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala
His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225
230 235 240 Arg Val Phe Lys Val
Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245
250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe
Gly Gly His Asp Ala Lys 260 265
270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr
Asn 275 280 285 Lys
Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290
295 300 Gly Thr Thr Ala Ser Leu
Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310
315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe
Ser Val Asp Lys Leu 325 330
335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp
340 345 350 Asn Phe Val
Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355
360 365 Phe Asp Lys Ala Val Phe Lys Ile
Asn Ile Val Pro Lys Val Asn Tyr 370 375
380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu
Ala Ala Asn385 390 395
400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu
405 410 415 Lys Asn Phe Thr
Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420
425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser
Leu Asp Lys Gly Tyr Asn Lys 435 440
445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu
Phe Phe 450 455 460
Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465
470 475 480 Ile Thr Ser Asp Thr
Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485
490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe
Asn Phe Asp Asn Glu Pro 500 505
510 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln
Leu 515 520 525 Glu
Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530
535 540 Leu Asp Lys Tyr Thr Met
Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550
555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser
Val Asn Glu Ala Leu 565 570
575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys
580 585 590 Lys Val Asn
Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595
600 605 Gln Leu Val Tyr Asp Phe Thr Asp
Glu Thr Ser Glu Val Ser Thr Thr 610 615
620 Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile
Gly Pro Ala625 630 635
640 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
645 650 655 Ile Phe Ser Gly
Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660
665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu
Val Ser Tyr Ile Ala Asn Lys 675 680
685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg
Asn Glu 690 695 700
Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705
710 715 720 Val Asn Thr Gln Ile
Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725
730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile
Ile Asn Tyr Gln Tyr Asn 740 745
750 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp
Asp 755 760 765 Leu
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770
775 780 Asn Lys Phe Leu Asn Gln
Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790
795 800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe
Asp Ala Ser Leu Lys 805 810
815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly
820 825 830 Gln Val Asp
Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835
840 845 Ile Pro Phe Gln Leu Ser Lys Tyr
Val Asp Asn Gln Arg Leu Leu Ser 850 855
860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser
Ile Leu Asn865 870 875
880 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser
885 890 895 Lys Ile Asn Ile
Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900
905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser
Ser Lys Ile Glu Val Ile Leu 915 920
925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser
Thr Ser 930 935 940
Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn945
950 955 960 Glu Tyr Thr Ile Ile
Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965
970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr
Leu Gln Asp Thr Gln Glu 980 985
990 Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile
Ser 995 1000 1005 Asp
Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010
1015 1020 Asn Asn Ser Lys Ile Tyr
Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025 1030
1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn
Asn Ile Met Phe Lys 1045 1050
1055 Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe
1060 1065 1070 Asn Leu Phe
Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075
1080 1085 Asp Asn Gln Ser Asn Ser Gly Ile
Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095
1100 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr
Asp Pro Asn1105 1110 1115
1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu
1125 1130 1135 Lys Gly Pro Arg
Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140
1145 1150 Ser Leu Tyr Arg Gly Thr Lys Phe Ile
Ile Lys Lys Tyr Ala Ser Gly 1155 1160
1165 Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile
Asn Val 1170 1175 1180
Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185
1190 1195 1200Gly Val Glu Lys Ile
Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn 1205
1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys
Asn Asp Gln Gly Ile Thr 1220 1225
1230 Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile
Gly 1235 1240 1245 Phe
Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250
1255 1260 Asn Trp Tyr Asn Arg Gln
Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265 1270
1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp
Gly Glu Arg Pro Leu 1285 1290
1295 21291PRTClostridium botulinum Serotype B 2 Met Pro Val Thr Ile
Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn1 5
10 15 Asn Asn Ile Ile Met Met Glu Pro Pro Phe
Ala Arg Gly Thr Gly Arg 20 25
30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro
Glu 35 40 45 Arg
Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50
55 60 Ile Phe Asn Arg Asp Val
Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70
75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr
Met Ile Lys Leu Phe 85 90
95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile
100 105 110 Ile Asn Gly
Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115
120 125 Phe Asn Thr Asn Ile Ala Ser Val
Thr Val Asn Lys Leu Ile Ser Asn 130 135
140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn
Leu Ile Ile145 150 155
160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly
165 170 175 Ile Gln Asn His
Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180
185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser
Val Phe Asn Asn Val Gln Glu 195 200
205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser
Asp Pro 210 215 220
Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225
230 235 240 Gly Ile Lys Val Asp
Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245
250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala
Glu Glu Leu Tyr Thr Phe 260 265
270 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser
Ile 275 280 285 Tyr
Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290
295 300 Lys Val Leu Val Cys Ile
Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310
315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val
Glu Asp Ser Glu Gly 325 330
335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu
340 345 350 Met Phe Gly
Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355
360 365 Thr Arg Ala Ser Tyr Phe Ser Asp
Ser Leu Pro Pro Val Lys Ile Lys 370 375
380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly
Phe Asn Ile385 390 395
400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile
405 410 415 Asn Lys Gln Ala
Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420
425 430 Lys Ile Gln Met Cys Lys Ser Val Lys
Ala Pro Gly Ile Cys Ile Asp 435 440
445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser
Phe Ser 450 455 460
Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn465
470 475 480 Tyr Ile Glu Asn Asp
Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485
490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu
Asn Thr Glu Ser Leu Thr 500 505
510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile
Lys 515 520 525 Lys
Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530
535 540 Thr Phe Pro Leu Asp Ile
Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp545 550
555 560 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser
Phe Phe Ser Met Asp 565 570
575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly
580 585 590 Trp Val Lys
Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595
600 605 Asn Thr Met Asp Lys Ile Ala Asp
Ile Ser Leu Ile Val Pro Tyr Ile 610 615
620 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly
Asn Phe Glu625 630 635
640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro
645 650 655 Glu Leu Leu Ile
Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660
665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr
Ile Asp Asn Ala Leu Thr Lys 675 680
685 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala
Gln Trp 690 695 700
Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr705
710 715 720 Lys Ala Leu Asn Tyr
Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725
730 735 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys
Ser Asn Ile Asn Ile Asp 740 745
750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala
Ile 755 760 765 Asp
Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770
775 780 Lys Lys Met Ile Pro Leu
Ala Val Glu Lys Leu Leu Asp Phe Asp Asn785 790
795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp
Glu Asn Lys Leu Tyr 805 810
815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu
820 825 830 Lys Thr Ile
Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835
840 845 Leu Ile Glu Met Phe Asn Lys Tyr
Asn Ser Glu Ile Leu Asn Asn Ile 850 855
860 Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp
Leu Ser Gly865 870 875
880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys
885 890 895 Asn Gln Phe Lys
Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900
905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser
Val Phe Leu Asp Phe Ser Val 915 920
925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile
Gln Asn 930 935 940
Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser945
950 955 960 Gly Trp Lys Ile Ser
Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965
970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe
Phe Glu Tyr Asn Ile Arg 980 985
990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile
Thr 995 1000 1005 Asn
Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010
1015 1020 Asn Thr Asp Ile Lys Asp
Ile Arg Glu Val Ile Ala Asn Gly Glu Ile1025 1030
1035 1040Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr
Gln Phe Ile Trp Met 1045 1050
1055 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu
1060 1065 1070 Glu Arg Tyr
Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075
1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys
Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095
1100 Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro
Val Gly Glu1105 1110 1115
1120Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr
1125 1130 1135 Arg Asp Leu Tyr
Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140
1145 1150 Ser Gln Ser Ile Asn Asp Asp Ile Val
Arg Lys Glu Asp Tyr Ile Tyr 1155 1160
1165 Leu Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr
Tyr Lys 1170 1175 1180
Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp1185
1190 1195 1200Ser Asp Glu Phe Tyr
Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln 1205
1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys
Lys Asp Glu Glu Ser Thr 1220 1225
1230 Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly
Ile 1235 1240 1245 Val
Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250
1255 1260 Lys Glu Val Lys Arg Lys
Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp1265 1270
1275 1280Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu
1285 1290 31291PRTClostridium botulinum
Serotype C1 3Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp
Asn1 5 10 15 Lys
Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20
25 30 Pro Glu Lys Ala Phe Arg
Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40
45 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn
Lys Pro Pro Arg Val 50 55 60
Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr
Asp65 70 75 80 Ser
Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg
85 90 95 Ile Asn Ser Arg Glu Ile
Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100
105 110 Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr
Pro Ile Asn Thr Phe Asp 115 120
125 Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln
Gly Asn 130 135 140
Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145
150 155 160 Pro Arg Glu Asn Ile
Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165
170 175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe
Gly Ala Leu Ser Ile Ile 180 185
190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn
Asp 195 200 205 Val
Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210
215 220 Leu Ile Leu Met His Glu
Leu Asn His Ala Met His Asn Leu Tyr Gly225 230
235 240 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser
Val Thr Ser Asn Ile 245 250
255 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala
260 265 270 Phe Gly Gly
Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275
280 285 Phe Glu Glu Lys Ala Leu Asp Tyr
Tyr Arg Ser Ile Ala Lys Arg Leu 290 295
300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys
Tyr Ile Gly305 310 315
320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser
325 330 335 Ser Gly Glu Val
Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340
345 350 Glu Leu Thr Gln Ile Phe Thr Glu Phe
Asn Tyr Ala Lys Ile Tyr Asn 355 360
365 Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro
Val Thr 370 375 380
Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385
390 395 400 Ile Pro Lys Ser Asn
Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405
410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro
Glu Asn Met Leu Tyr Leu 420 425
430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr
Asn 435 440 445 Lys
Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450
455 460 Phe Ile Gly Asp Ile Ser
Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470
475 480 Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr
Pro Asp Asn Val Ser 485 490
495 Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu
500 505 510 Asp Leu Leu
Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515
520 525 Glu Asn Gln Val Phe Tyr Asp Asn
Arg Thr Gln Asn Val Asp Tyr Leu 530 535
540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp
Asn Val Glu545 550 555
560 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala
565 570 575 Lys Val Tyr Thr
Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580
585 590 Val Gln Gly Gly Leu Phe Leu Met Trp
Ala Asn Asp Val Val Glu Asp 595 600
605 Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile
Ser Asp 610 615 620
Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625
630 635 640 Ser Val Arg Arg Gly
Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645
650 655 Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe
Thr Ile Pro Ala Leu Gly 660 665
670 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile
Lys 675 680 685 Thr
Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690
695 700 Tyr Glu Trp Met Met Gly
Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710
715 720 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu
Asn Tyr Gln Ala Gly 725 730
735 Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser
740 745 750 Asp Lys Glu
Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755
760 765 Asp Val Lys Ile Ser Glu Ala Met
Asn Asn Ile Asn Lys Phe Ile Arg 770 775
780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro
Lys Val Ile785 790 795
800 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn
805 810 815 Leu Ile Asp Ser
His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820
825 830 Lys Ala Lys Val Asn Asn Ser Phe Gln
Asn Thr Ile Pro Phe Asn Ile 835 840
845 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn
Glu Tyr 850 855 860
Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys865
870 875 880 Asn Thr Leu Val Asp
Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885
890 895 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro
Phe Asp Phe Lys Leu Gly 900 905
910 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu
Asn 915 920 925 Ile
Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930
935 940 Arg Ile Asn Lys Trp Val
Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp945 950
955 960 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly
Ile Ile Ser Asn Phe 965 970
975 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn
980 985 990 Phe Ser Tyr
Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995
1000 1005 Phe Val Thr Val Thr Asn Asn Met
Met Gly Asn Met Lys Ile Tyr Ile 1010 1015
1020 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu
Thr Gly Ile1025 1030 1035
1040Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr
1045 1050 1055 Gly Leu Ile Thr
Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 1060
1065 1070 Phe Tyr Ile Phe Ala Lys Glu Leu Asp
Gly Lys Asp Ile Asn Ile Leu 1075 1080
1085 Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp
Gly Asn 1090 1095 1100
Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp Tyr Leu1105
1110 1115 1120Asn Arg Tyr Met Tyr
Ala Asn Ser Arg Gln Ile Val Phe Asn Thr Arg 1125
1130 1135 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr
Lys Ile Ile Ile Lys Arg 1140 1145
1150 Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile
Leu 1155 1160 1165 Tyr
Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 1170
1175 1180 Asn Glu Thr Met Tyr Ala
Asp Asn His Ser Thr Glu Asp Ile Tyr Ala1185 1190
1195 1200Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile Asn
Asp Asn Ile Ile Phe 1205 1210
1215 Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe
1220 1225 1230 Lys Ser Asn
Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235
1240 1245 Thr Tyr Arg Phe Arg Leu Gly Gly
Asp Trp Tyr Arg His Asn Tyr Leu 1250 1255
1260 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu
Glu Ser Thr1265 1270 1275
1280Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1285
1290 41276PRTClostridium botulinum Serotype D 4Met Thr Trp
Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5
10 15 Asn Asp Ile Leu Tyr Leu Arg Ile
Pro Gln Asn Lys Leu Ile Thr Thr 20 25
30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val
Ile Pro Glu 35 40 45
Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50
55 60 Thr Ser Lys Tyr Gln
Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp65 70
75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile
Ile Lys Leu Phe Lys Arg 85 90
95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val
Val 100 105 110 Gly
Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115
120 125 Phe Thr Arg His Thr Thr
Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135
140 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser
Val Leu Ile Phe Gly145 150 155
160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly
165 170 175 Gln Gln Ser
Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180
185 190 Lys Val Ala Pro Glu Phe Leu Leu
Thr Phe Ser Asp Val Thr Ser Asn 195 200
205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met
Asp Pro Val 210 215 220
Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly225
230 235 240 Ile Asn Ile Pro Ser
Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245
250 255 Phe Phe Ser Gln Asp Gly Pro Asn Val Gln
Phe Glu Glu Leu Tyr Thr 260 265
270 Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser
Gln 275 280 285 Leu
Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290
295 300 Asn Asn Ile Asn Lys Thr
Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp305 310
315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn
Phe Asp Lys Asp Asn 325 330
335 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser
340 345 350 Asp Leu Thr
Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355
360 365 Val Lys Asn Arg Thr His Tyr Phe
Ser Arg His Tyr Leu Pro Val Phe 370 375
380 Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp
Gly Phe Asn385 390 395
400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu
405 410 415 Arg Asn Pro Ala
Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420
425 430 Phe Thr Lys Val Cys Leu Arg Leu Thr
Lys Asn Ser Arg Asp Asp Ser 435 440
445 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala
Asp Lys 450 455 460
Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu465
470 475 480 Thr Asn Val Gln Asn
Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile 485
490 495 Leu Asp Gly Gln Val Pro Ile Asn Pro Glu
Ile Val Asp Pro Leu Leu 500 505
510 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile
Val 515 520 525 Phe
Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530
535 540 Tyr Leu Glu Ser Gln Lys
Leu Ser Asn Asn Val Glu Asn Ile Thr Leu545 550
555 560 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser
Asn Lys Ile Tyr Thr 565 570
575 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly
580 585 590 Leu Phe Leu
Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595
600 605 Ile Met Lys Lys Asp Thr Leu Asp
Lys Ile Ser Asp Val Ser Val Ile 610 615
620 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser
Ala Leu Arg625 630 635
640 Gly Asn Phe Asn Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu
645 650 655 Glu Gly Phe Pro
Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660
665 670 Tyr Ser Ser Ile Gln Glu Arg Glu Lys
Ile Ile Lys Thr Ile Glu Asn 675 680
685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln
Trp Met 690 695 700
Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn705
710 715 720 Tyr Gln Met Tyr Asp
Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725
730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser
Gly Ser Asp Lys Glu Asn 740 745
750 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys
Ile 755 760 765 Ser
Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770
775 780 Thr Tyr Leu Phe Lys Asn
Met Leu Pro Lys Val Ile Asp Glu Leu Asn785 790
795 800 Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile
Asn Leu Ile Asp Ser 805 810
815 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val
820 825 830 Asn Glu Ser
Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835
840 845 Asn Asn Ser Leu Leu Lys Asp Ile
Ile Asn Glu Tyr Phe Asn Ser Ile 850 855
860 Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn
Ala Leu Val865 870 875
880 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln
885 890 895 Leu Asn Thr Ile
Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900
905 910 Lys Ile Ile Val Asn Leu Asn Asn Asn
Ile Leu Tyr Ser Ala Ile Tyr 915 920
925 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp
Leu Thr 930 935 940
Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser945
950 955 960 Gly Trp Lys Leu Cys
Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965
970 975 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile
Phe Asp Tyr Ser Glu Ser 980 985
990 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile
Thr 995 1000 1005 Asn
Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys 1010
1015 1020 Gln Ser Gln Lys Ile Glu
Asp Leu Asp Glu Val Lys Leu Asp Lys Thr1025 1030
1035 1040Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu
Asn Gln Met Leu Trp 1045 1050
1055 Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser Asn Glu Asp Ile
1060 1065 1070 Asn Ile Val
Tyr Glu Gly Gln Ile Leu Arg Asn Val Ile Lys Asp Tyr 1075
1080 1085 Trp Gly Asn Pro Leu Lys Phe Asp
Thr Glu Tyr Tyr Ile Ile Asn Asp 1090 1095
1100 Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn Val
Leu Val Leu1105 1110 1115
1120Val Gln Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro Ile Thr
1125 1130 1135 Ile Lys Ser Val
Ser Asp Lys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140
1145 1150 Asp Asn Ile Ile Leu His Met Leu Tyr
Asn Ser Arg Lys Tyr Met Ile 1155 1160
1165 Ile Arg Asp Thr Asp Thr Ile Tyr Ala Thr Gln Gly Gly Glu
Cys Ser 1170 1175 1180
Gln Asn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr1185
1190 1195 1200Gly Ile Gly Ile Phe
Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr 1205
1210 1215 Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu
Asn Thr Met Leu Leu Ala 1220 1225
1230 Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr
Pro 1235 1240 1245 Val
Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250
1255 1260 Trp Lys Phe Ile Ser Arg
Asp Pro Gly Trp Val Glu1265 1270 1275
51252PRTClostridium botulinum Serotype E 5Met Pro Lys Ile Asn Ser Phe Asn
Tyr Asn Asp Pro Val Asn Asp Arg1 5 10
15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe
Tyr Lys Ser 20 25 30
Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile
35 40 45 Gly Thr Thr Pro
Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55
60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr
Leu Gln Ser Asp Glu Glu Lys65 70 75
80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile
Asn Asn 85 90 95
Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro
100 105 110 Tyr Leu Gly Asn Asp
Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115
120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser
Asn Gly Ser Gln Asp Ile Leu 130 135
140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu
Phe Glu Thr145 150 155
160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His
165 170 175 Gly Phe Gly Ser
Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180
185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu
Phe Ile Gln Asp Pro Ala Leu 195 200
205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr
Gly Ala 210 215 220
Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225
230 235 240 Ile Thr Asn Ile Arg
Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245
250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala
Gln Ser Asn Asp Ile Tyr 260 265
270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser
Lys 275 280 285 Val
Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290
295 300 Ala Lys Tyr Gly Leu Asp
Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310
315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu
Tyr Ser Phe Thr Glu 325 330
335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile
340 345 350 Gly Gln Tyr
Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355
360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn
Ile Asn Asn Leu Lys Val Asn Phe 370 375
380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr
Pro Ile Thr385 390 395
400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val
405 410 415 Ser Val Lys Gly
Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420
425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn
Ser Tyr Asn Asp Asp Asn Ile 435 440
445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn
Asn Tyr 450 455 460
Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465
470 475 480 Pro Gly Leu Ser Asp
Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485
490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr
Ser Asp Ile Glu Gln His 500 505
510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys
Val 515 520 525 Pro
Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530
535 540 Leu Leu Glu Gln Pro Lys
Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550
555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu
Phe Val Ser Trp Ile 565 570
575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr
580 585 590 Val Asp Lys
Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595
600 605 Ala Leu Asn Ile Gly Asn Glu Ala
Gln Lys Gly Asn Phe Lys Asp Ala 610 615
620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu
Pro Glu Leu625 630 635
640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser
645 650 655 Ser Asp Asn Lys
Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660
665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val
Tyr Ser Phe Ile Val Ser Asn 675 680
685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu
Gln Met 690 695 700
Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu705
710 715 720 Ser Lys Tyr Asn Ser
Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725
730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu
Leu Asn Gln Lys Val Ser 740 745
750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile
Ser 755 760 765 Tyr
Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770
775 780 Tyr Asp Glu Asn Val Lys
Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His785 790
795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu
Asn Ser Met Val Thr 805 810
815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp
820 825 830 Asp Lys Ile
Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835
840 845 Ser Ser Ser Val Leu Asn Met Arg
Tyr Lys Asn Asp Lys Tyr Val Asp 850 855
860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp
Val Tyr Lys865 870 875
880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser
885 890 895 Glu Val Asn Ile
Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900
905 910 Lys Asn Phe Ser Ile Ser Phe Trp Val
Arg Ile Pro Asn Tyr Asp Asn 915 920
925 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys
Met Arg 930 935 940
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile945
950 955 960 Trp Thr Leu Gln Asp
Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965
970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe 980 985
990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile
Asn 995 1000 1005 Gly
Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010
1015 1020 Val Ser Asp Asn Ile Leu
Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025 1030
1035 1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp
Lys Glu Leu Asp Glu 1045 1050
1055 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu
1060 1065 1070 Lys Asp Phe
Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075
1080 1085 Leu Asn Val Leu Lys Pro Asn Asn
Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095
1100 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu
Ala Asn Arg1105 1110 1115
1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser
1125 1130 1135 Thr Asn Asp Asn
Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140
1145 1150 Val Ala Ser Lys Thr His Leu Phe Pro
Leu Tyr Ala Asp Thr Ala Thr 1155 1160
1165 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn
Arg Phe 1170 1175 1180
Asn Gln Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn1185
1190 1195 1200Phe Lys Asn Asn Asn
Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205
1210 1215 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr
Thr His Met Arg Asp His 1220 1225
1230 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His
Gly 1235 1240 1245 Trp
Gln Glu Lys 1250 61274PRTClostridium botulinum Serotype F 6Met
Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp1
5 10 15 Asp Thr Ile Leu Tyr Met
Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25
30 Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val
Trp Ile Ile Pro Glu 35 40 45
Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser
50 55 60 Leu Lys Asn
Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65 70
75 80 Asp Ala Glu Lys Asp Arg Tyr Leu
Lys Thr Thr Ile Lys Leu Phe Lys 85 90
95 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln
Glu Ile Ser 100 105 110
Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe
115 120 125 Ser Pro Val Thr
Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130
135 140 Val Glu Ser Ser Met Leu Leu Asn
Leu Leu Val Leu Gly Ala Gly Pro145 150
155 160 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys
Leu Ile Asp Pro 165 170
175 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile
180 185 190 Val Thr Phe
Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195
200 205 Gly His Asn Ser Ser Thr Glu Ser
Phe Ile Ala Asp Pro Ala Ile Ser 210 215
220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr
Gly Ala Arg225 230 235
240 Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala Pro Leu Met
245 250 255 Ile Ala Glu Lys
Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260
265 270 Gln Asp Leu Asn Ile Ile Thr Ser Ala
Met Lys Glu Lys Ile Tyr Asn 275 280
285 Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser
Glu Val 290 295 300
Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe305
310 315 320 Gln Trp Lys Tyr Gly
Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325
330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys
Lys Leu Tyr Ser Phe Thr 340 345
350 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr
Tyr 355 360 365 Phe
Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370
375 380 Ile Tyr Thr Val Ser Glu
Gly Phe Asn Ile Gly Asn Leu Ala Val Asn385 390
395 400 Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys
Ile Ile Asp Ser Ile 405 410
415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val
420 425 430 Ile Pro Arg
Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435
440 445 Asn Asn Ser Glu Leu Phe Phe Val
Ala Ser Glu Ser Ser Tyr Asn Glu 450 455
460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr
Asn Leu Asn465 470 475
480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser
485 490 495 Gln Thr Ile Pro
Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gln 500
505 510 Asp Asn Ser Tyr Val Pro Arg Tyr Asp
Ser Asn Gly Thr Ser Glu Ile 515 520
525 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu
His Ala 530 535 540
Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile545
550 555 560 Asp Thr Ala Leu Leu
Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565
570 575 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn
Ala Ala Leu Phe Ile Asp 580 585
590 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln
Lys 595 600 605 Ser
Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610
615 620 Gly Leu Ala Leu Asn Ile
Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu625 630
635 640 Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu
Leu Glu Phe Val Pro 645 650
655 Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile
660 665 670 Asp Ser Tyr
Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675
680 685 Leu Ile Glu Arg Glu Ala Lys Trp
Lys Glu Ile Tyr Ser Trp Ile Val 690 695
700 Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys
Arg Lys Glu705 710 715
720 Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala
725 730 735 Ile Glu Tyr Lys
Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740
745 750 Glu Ser Glu Tyr Asn Ile Asn Asn Ile
Glu Glu Glu Leu Asn Lys Lys 755 760
765 Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu
Ser Ser 770 775 780
Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu785
790 795 800 Lys Lys Tyr Asp Asn
His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805
810 815 Asp His Arg Ser Ile Leu Gly Glu Gln Thr
Asn Glu Leu Ser Asp Leu 820 825
830 Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser
Tyr 835 840 845 Thr
Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850
855 860 Ile Lys Asp Ser Ser Ile
Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe865 870
875 880 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser
Ile Asn Gly Asn Val 885 890
895 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Asn Ser Arg
900 905 910 Leu Ser Glu
Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Ser 915
920 925 Arg Tyr Gln Asn Phe Ser Ile Ser
Phe Trp Val Arg Ile Pro Lys His 930 935
940 Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile
Asn Cys Met945 950 955
960 Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp
965 970 975 Cys Glu Ile Ile
Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980
985 990 Leu Ile Phe Arg Tyr Glu Glu Leu Asn
Arg Ile Ser Asn Tyr Ile Asn 995 1000
1005 Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn
Ser Arg 1010 1015 1020
Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu1025
1030 1035 1040Gly Asp Ile His Val
Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 1045
1050 1055 Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr
Phe Lys Val Phe Asn Thr 1060 1065
1070 Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro
Asp 1075 1080 1085 Pro
Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 1090
1095 1100 Lys Tyr Tyr Leu Phe Asn
Leu Leu Arg Lys Asp Lys Tyr Ile Thr Leu1105 1110
1115 1120Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg
Gly Val Thr Glu Gly 1125 1130
1135 Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val Ile Ile
1140 1145 1150 Arg Lys Asn
Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe Val Arg 1155
1160 1165 Lys Asn Asp Leu Ala Tyr Ile Asn
Val Val Asp Arg Gly Val Glu Tyr 1170 1175
1180 Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile
Ile Arg Thr1185 1190 1195
1200Ser Asn Leu Asn Asp Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile
1205 1210 1215 Gly Asn Asn Cys
Thr Met Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile 1220
1225 1230 Gly Leu Leu Gly Phe His Ser Asn Asn
Leu Val Ala Ser Ser Trp Tyr 1235 1240
1245 Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe
Trp Ser 1250 1255 1260
Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu1265 1270
71297PRTClostridium botulinum Serotype G 7Met Pro Val Asn Ile Lys
Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn1 5
10 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp
Pro Gly Pro Gly Thr 20 25 30
Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu
35 40 45 Arg Phe Thr
Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50
55 60 Val Phe Ser Lys Asp Val Tyr Glu
Tyr Tyr Asp Pro Thr Tyr Leu Lys65 70 75
80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile
Lys Leu Phe 85 90 95
Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile
100 105 110 Val Asp Ala Ile Pro
Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115
120 125 Phe Ala Ala Asn Val Ala Asn Val Ser
Ile Asn Lys Lys Ile Ile Gln 130 135
140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn
Leu Ile Ile145 150 155
160 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile
165 170 175 Met Asn Gly His
Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180
185 190 Ile Arg Phe Cys Pro Ser Cys Leu Asn
Val Phe Asn Asn Val Gln Glu 195 200
205 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala
Asp Pro 210 215 220
Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225
230 235 240 Gly Ile Lys Ile Ser
Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245
250 255 Phe Met Gln His Ser Asp Pro Val Gln Ala
Glu Glu Leu Tyr Thr Phe 260 265
270 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn
Ile 275 280 285 Tyr
Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290
295 300 Ile Val Ser Ser Ala Gln
Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys305 310
315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu
Asp Pro Asn Gly Lys 325 330
335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met
340 345 350 Phe Gly Phe
Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr 355
360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr
Leu Pro Pro Ile Lys Thr Glu Lys 370 375
380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe
Asn Ile Ala385 390 395
400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn
405 410 415 Lys Glu Ala Tyr
Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420
425 430 Ile Ala Met Cys Lys Pro Val Met Tyr
Lys Asn Thr Gly Lys Ser Glu 435 440
445 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala
Asn Lys 450 455 460
Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn465
470 475 480 Thr Gln Asn Asn Thr
Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485
490 495 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp
Leu Pro Asn Glu Asn Thr 500 505
510 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile
Lys 515 520 525 Gln
Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530
535 540 Tyr Leu His Ala Gln Thr
Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu545 550
555 560 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn
Asn Lys Val Tyr Thr 565 570
575 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala
580 585 590 Ser Leu Phe
Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser 595
600 605 Glu Ser Thr Gln Lys Ser Thr Ile
Asp Lys Val Ser Asp Val Ser Ile 610 615
620 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn
Glu Thr Ala625 630 635
640 Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu
645 650 655 Met Glu Phe Ile
Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr 660
665 670 Leu Glu Ser Tyr Val Gly Asn Lys Gly
His Ile Ile Met Thr Ile Ser 675 680
685 Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr
Gly Leu 690 695 700
Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile705
710 715 720 Lys Glu Arg Met Tyr
Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725
730 735 Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr
Ser Glu Glu Asp Lys Met 740 745
750 Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln
Ser 755 760 765 Ile
Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770
775 780 Ile Ser Tyr Leu Met Asn
Arg Met Ile Pro Leu Ala Val Lys Lys Leu785 790
795 800 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu
Leu Glu Tyr Ile Asp 805 810
815 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys
820 825 830 Val Asn Arg
His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835
840 845 Thr Lys Asp Thr Ile Leu Ile Gln
Val Phe Asn Asn Tyr Ile Ser Asn 850 855
860 Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly
Gly Arg Leu865 870 875
880 Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val
885 890 895 Ile Phe Asn Asp
Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu 900
905 910 Asn Ser Asn Ile Thr Ala His Gln Ser
Lys Phe Val Val Tyr Asp Ser 915 920
925 Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro
Lys Tyr 930 935 940
Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile945
950 955 960 Ser Cys Ile Lys Asn
Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965
970 975 Arg Ile Ile Trp Thr Leu Ile Asp Val Asn
Ala Lys Ser Lys Ser Ile 980 985
990 Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn
Lys 995 1000 1005 Trp
Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile 1010
1015 1020 Tyr Ile Asn Gly Ser Leu
Lys Lys Ser Glu Lys Ile Leu Asn Leu Asp1025 1030
1035 1040Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys
Leu Ile Asn Cys Thr 1045 1050
1055 Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg
1060 1065 1070 Glu Leu Asn
Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser 1075
1080 1085 Thr Asn Thr Leu Lys Asp Phe Trp
Gly Asn Pro Leu Arg Tyr Asp Thr 1090 1095
1100 Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn Ile Tyr
Ile Lys Tyr1105 1110 1115
1120Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn
1125 1130 1135 Asn Ala Ala Ile
Asn Tyr Gln Asn Leu Tyr Leu Gly Leu Arg Phe Ile 1140
1145 1150 Ile Lys Lys Ala Ser Asn Ser Arg Asn
Ile Asn Asn Asp Asn Ile Val 1155 1160
1165 Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser
Asp Glu 1170 1175 1180
Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln1185
1190 1195 1200Leu Phe Leu Ala Pro
Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205
1210 1215 Gln Ile Lys Lys Tyr Tyr Glu Lys Thr Thr
Tyr Asn Cys Gln Ile Leu 1220 1225
1230 Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys
Phe 1235 1240 1245 Val
Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250
1255 1260 Ile Ser Gln Trp Tyr Leu
Arg Arg Ile Ser Glu Asn Ile Asn Lys Leu1265 1270
1275 1280Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val
Asp Glu Gly Trp Thr 1285 1290
1295 Glu81315PRTClostridium tetani 8Met Pro Ile Thr Ile Asn Asn Phe
Arg Tyr Ser Asp Pro Val Asn Asn1 5 10
15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly
Leu Asp Ile 20 25 30
Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu
35 40 45 Arg Tyr Glu Phe
Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55
60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr
Asp Pro Asn Tyr Leu Arg Thr65 70 75
80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu
Phe Asn 85 90 95
Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile
100 105 110 Asn Ala Ile Pro Tyr
Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115
120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe
Asn Leu Leu Glu Gln Asp Pro 130 135
140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu
Ile Ile Phe145 150 155
160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu
165 170 175 Arg Val Asp Asn
Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180
185 190 Ile Met Gln Met Ala Phe Cys Pro Glu
Tyr Val Pro Thr Phe Asp Asn 195 200
205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys
Tyr Phe 210 215 220
Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His225
230 235 240 Gly Leu Tyr Gly Met
Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245
250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro
Ile Ser Ala Glu Glu Leu 260 265
270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile
Lys 275 280 285 Asn
Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290
295 300 Lys Leu Ser Gln Val Thr
Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp305 310
315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln
Phe Asp Lys Asp Ser 325 330
335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn
340 345 350 Ser Ile Met
Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355
360 365 Ile Lys Thr Arg Leu Ser Tyr Phe
Ser Met Asn His Asp Pro Val Lys 370 375
380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr
Glu Gly Phe385 390 395
400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met
405 410 415 Arg Val Asn Thr
Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420
425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys
Ile Ile Pro Pro Thr Asn Ile 435 440
445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu
Gly Gly 450 455 460
Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu465
470 475 480 Lys Asn Ser Phe Ser
Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485
490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn
Tyr Ser Leu Asp Lys Ile 500 505
510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp
Arg 515 520 525 Thr
Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530
535 540 Asn Ala Ala Ser Thr Ile
Glu Ile His Asn Ile Asp Asp Asn Thr Ile545 550
555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr
Thr Leu Gln Arg Ile 565 570
575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile
580 585 590 Tyr Ser Tyr
Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595
600 605 Gly Ile Leu Phe Leu Gln Trp Val
Arg Asp Ile Ile Asp Asp Phe Thr 610 615
620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser
Asp Val Ser625 630 635
640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly
645 650 655 Tyr Glu Gly Asn
Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660
665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr
Leu Pro Val Ile Ala Ala Leu 675 680
685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys
Thr Ile 690 695 700
Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys705
710 715 720 Leu Val Lys Ala Lys
Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725
730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu
Tyr Gln Val Asp Ala Ile 740 745
750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp
Lys 755 760 765 Glu
Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770
775 780 Lys Ala Asn Lys Ala Met
Ile Asn Ile Asn Ile Phe Met Arg Glu Ser785 790
795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn
Glu Ala Lys Lys Gln 805 810
815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile
820 825 830 Lys Ala Asn
Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835
840 845 Ser Lys Ile Asn Lys Val Phe Ser
Thr Pro Ile Pro Phe Ser Tyr Ser 850 855
860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile
Asp Val Ile865 870 875
880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile
885 890 895 Ser Asp Ile Ser
Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900
905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys
Ala Ile His Leu Val Asn Asn 915 920
925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu
Tyr Asn 930 935 940
Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys945
950 955 960 Val Ser Ala Ser His
Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965
970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser
Ile Gly Ser Gly Trp Ser 980 985
990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser
Ala 995 1000 1005 Gly
Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010
1015 1020 Ala Tyr Leu Ala Asn Lys
Trp Val Phe Ile Thr Ile Thr Asn Asp Arg1025 1030
1035 1040Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val
Leu Met Gly Ser Ala 1045 1050
1055 Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu
1060 1065 1070 Lys Leu Asp
Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys 1075
1080 1085 Phe Arg Ile Phe Cys Lys Ala Leu
Asn Pro Lys Glu Ile Glu Lys Leu 1090 1095
1100 Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe
Trp Gly Asn1105 1110 1115
1120Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser
1125 1130 1135 Ser Lys Asp Val
Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 1140
1145 1150 Asn Ala Pro Ser Tyr Thr Asn Gly Lys
Leu Asn Ile Tyr Tyr Arg Arg 1155 1160
1165 Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro
Asn Asn 1170 1175 1180
Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val1185
1190 1195 1200Ser Tyr Asn Asn Asn
Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn 1205
1210 1215 Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg
Val Gly Tyr Asn Ala Pro 1220 1225
1230 Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp
Leu 1235 1240 1245 Lys
Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250
1255 1260 Leu Gly Leu Val Gly Thr
His Asn Gly Gln Ile Gly Asn Asp Pro Asn1265 1270
1275 1280Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe
Asn His Leu Lys Asp 1285 1290
1295 Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp
1300 1305 1310 Thr Asn Asp
131591268PRTClostridium baratii 9Met Pro Val Asn Ile Asn Asn Phe
Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10
15 Thr Thr Ile Leu Tyr Met Lys Met Pro Tyr Tyr Glu Asp
Ser Asn Lys 20 25 30
Tyr Tyr Lys Ala Phe Glu Ile Met Asp Asn Val Trp Ile Ile Pro Glu
35 40 45 Arg Asn Ile Ile
Gly Lys Lys Pro Ser Asp Phe Tyr Pro Pro Ile Ser 50 55
60 Leu Asp Ser Gly Ser Ser Ala Tyr Tyr
Asp Pro Asn Tyr Leu Thr Thr65 70 75
80 Asp Ala Glu Lys Asp Arg Phe Leu Lys Thr Val Ile Lys Leu
Phe Asn 85 90 95
Arg Ile Asn Ser Asn Pro Ala Gly Gln Val Leu Leu Glu Glu Ile Lys
100 105 110 Asn Gly Lys Pro Tyr
Leu Gly Asn Asp His Thr Ala Val Asn Glu Phe 115
120 125 Cys Ala Asn Asn Arg Ser Thr Ser Val
Glu Ile Lys Glu Ser Asn Gly 130 135
140 Thr Thr Asp Ser Met Leu Leu Asn Leu Val Ile Leu Gly
Pro Gly Pro145 150 155
160 Asn Ile Leu Glu Cys Ser Thr Phe Pro Val Arg Ile Phe Pro Asn Asn
165 170 175 Ile Ala Tyr Asp
Pro Ser Glu Lys Gly Phe Gly Ser Ile Gln Leu Met 180
185 190 Ser Phe Ser Thr Glu Tyr Glu Tyr Ala
Phe Asn Asp Asn Thr Asp Leu 195 200
205 Phe Ile Ala Asp Pro Ala Ile Ser Leu Ala His Glu Leu Ile
His Val 210 215 220
Leu His Gly Leu Tyr Gly Ala Lys Gly Val Thr Asn Lys Lys Val Ile225
230 235 240 Glu Val Asp Gln Gly
Ala Leu Met Ala Ala Glu Lys Asp Ile Lys Ile 245
250 255 Glu Glu Phe Ile Thr Phe Gly Gly Gln Asp
Leu Asn Ile Ile Thr Asn 260 265
270 Ser Thr Asn Gln Lys Ile Tyr Val Ile Leu Leu Ser Asn Tyr Thr
Ala 275 280 285 Ile
Ala Ser Arg Leu Ser Gln Val Asn Arg Asn Asn Ser Ala Leu Asn 290
295 300 Thr Thr Tyr Tyr Lys Asn
Phe Phe Gln Trp Lys Tyr Gly Leu Asp Gln305 310
315 320 Asp Ser Asn Gly Asn Tyr Thr Val Asn Ile Ser
Lys Phe Asn Ala Ile 325 330
335 Tyr Lys Lys Leu Phe Ser Phe Thr Glu Cys Asp Leu Ala Gln Lys Phe
340 345 350 Gln Val Lys
Asn Arg Ser Asn Tyr Leu Phe His Phe Lys Pro Phe Arg 355
360 365 Leu Leu Asp Leu Leu Asp Asp Asn
Ile Tyr Ser Ile Ser Glu Gly Phe 370 375
380 Asn Ile Gly Ser Leu Arg Val Asn Asn Asn Gly Gln Asn
Ile Asn Leu385 390 395
400 Asn Ser Arg Ile Val Gly Pro Ile Pro Asp Asn Gly Leu Val Glu Arg
405 410 415 Phe Val Gly Leu
Cys Lys Ser Ile Val Ser Lys Lys Gly Thr Lys Asn 420
425 430 Ser Leu Cys Ile Lys Val Asn Asn Arg
Asp Leu Phe Phe Val Ala Ser 435 440
445 Glu Ser Ser Tyr Asn Glu Asn Gly Ile Asn Ser Pro Lys Glu
Ile Asp 450 455 460
Asp Thr Thr Ile Thr Asn Asn Asn Tyr Lys Lys Asn Leu Asp Glu Val465
470 475 480 Ile Leu Asp Tyr Asn
Ser Asp Ala Ile Pro Asn Leu Ser Ser Arg Leu 485
490 495 Leu Asn Thr Thr Ala Gln Asn Asp Ser Tyr
Val Pro Lys Tyr Asp Ser 500 505
510 Asn Gly Thr Ser Glu Ile Lys Glu Tyr Thr Val Asp Lys Leu Asn
Val 515 520 525 Phe
Phe Tyr Leu Tyr Ala Gln Lys Ala Pro Glu Gly Glu Ser Ala Ile 530
535 540 Ser Leu Thr Ser Ser Val
Asn Thr Ala Leu Leu Asp Ala Ser Lys Val545 550
555 560 Tyr Thr Phe Phe Ser Ser Asp Phe Ile Asn Thr
Val Asn Lys Pro Val 565 570
575 Gln Ala Ala Leu Phe Ile Ser Trp Ile Gln Gln Val Ile Asn Asp Phe
580 585 590 Thr Thr Glu
Ala Thr Gln Lys Ser Thr Ile Asp Lys Ile Ala Asp Ile 595
600 605 Ser Leu Ile Val Pro Tyr Val Gly
Leu Ala Leu Asn Ile Gly Asn Glu 610 615
620 Val Gln Lys Gly Asn Phe Lys Glu Ala Ile Glu Leu Leu
Gly Ala Gly625 630 635
640 Ile Leu Leu Glu Phe Val Pro Glu Leu Leu Ile Pro Thr Ile Leu Val
645 650 655 Phe Thr Ile Lys
Ser Phe Ile Asn Ser Asp Asp Ser Lys Asn Lys Ile 660
665 670 Ile Lys Ala Ile Asn Asn Ala Leu Arg
Glu Arg Glu Leu Lys Trp Lys 675 680
685 Glu Val Tyr Ser Trp Ile Val Ser Asn Trp Leu Thr Arg Ile
Asn Thr 690 695 700
Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr Gln Ala Leu Gln Asn Gln705
710 715 720 Val Asp Gly Ile Lys
Lys Ile Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr 725
730 735 Leu Asp Glu Lys Asn Arg Leu Arg Ala Glu
Tyr Asn Ile Tyr Ser Ile 740 745
750 Lys Glu Glu Leu Asn Lys Lys Val Ser Leu Ala Met Gln Asn Ile
Asp 755 760 765 Arg
Phe Leu Thr Glu Ser Ser Ile Ser Tyr Leu Met Lys Leu Ile Asn 770
775 780 Glu Ala Lys Ile Asn Lys
Leu Ser Glu Tyr Asp Lys Arg Val Asn Gln785 790
795 800 Tyr Leu Leu Asn Tyr Ile Leu Glu Asn Ser Ser
Thr Leu Gly Thr Ser 805 810
815 Ser Val Pro Glu Leu Asn Asn Leu Val Ser Asn Thr Leu Asn Asn Ser
820 825 830 Ile Pro Phe
Glu Leu Ser Glu Tyr Thr Asn Asp Lys Ile Leu Ile His 835
840 845 Ile Leu Ile Arg Phe Tyr Lys Arg
Ile Ile Asp Ser Ser Ile Leu Asn 850 855
860 Met Lys Tyr Glu Asn Asn Arg Phe Ile Asp Ser Ser Gly
Tyr Gly Ser865 870 875
880 Asn Ile Ser Ile Asn Gly Asp Ile Tyr Ile Tyr Ser Thr Asn Arg Asn
885 890 895 Gln Phe Gly Ile
Tyr Ser Ser Arg Leu Ser Glu Val Asn Ile Thr Gln 900
905 910 Asn Asn Thr Ile Ile Tyr Asn Ser Arg
Tyr Gln Asn Phe Ser Val Ser 915 920
925 Phe Trp Val Arg Ile Pro Lys Tyr Asn Asn Leu Lys Asn Leu
Asn Asn 930 935 940
Glu Tyr Thr Ile Ile Asn Cys Met Arg Asn Asn Asn Ser Gly Trp Lys945
950 955 960 Ile Ser Leu Asn Tyr
Asn Asn Ile Ile Trp Thr Leu Gln Asp Thr Thr 965
970 975 Gly Asn Asn Gln Lys Leu Val Phe Asn Tyr
Thr Gln Met Ile Asp Ile 980 985
990 Ser Asp Tyr Ile Asn Lys Trp Thr Phe Val Thr Ile Thr Asn Asn
Arg 995 1000 1005 Leu
Gly His Ser Lys Leu Tyr Ile Asn Gly Asn Leu Thr Asp Gln Lys 1010
1015 1020 Ser Ile Leu Asn Leu Gly
Asn Ile His Val Asp Asp Asn Ile Leu Phe1025 1030
1035 1040Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val
Gly Ile Arg Tyr Phe 1045 1050
1055 Lys Ile Phe Asn Met Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr
1060 1065 1070 His Ser Glu
Pro Asp Ser Thr Ile Leu Lys Asp Phe Trp Gly Asn Tyr 1075
1080 1085 Leu Leu Tyr Asn Lys Lys Tyr Tyr
Leu Leu Asn Leu Leu Lys Pro Asn 1090 1095
1100 Met Ser Val Thr Lys Asn Ser Asp Ile Leu Asn Ile Asn
Arg Gln Arg1105 1110 1115
1120Gly Ile Tyr Ser Lys Thr Asn Ile Phe Ser Asn Ala Arg Leu Tyr Thr
1125 1130 1135 Gly Val Glu Val
Ile Ile Arg Lys Val Gly Ser Thr Asp Thr Ser Asn 1140
1145 1150 Thr Asp Asn Phe Val Arg Lys Asn Asp
Thr Val Tyr Ile Asn Val Val 1155 1160
1165 Asp Gly Asn Ser Glu Tyr Gln Leu Tyr Ala Asp Val Ser Thr
Ser Ala 1170 1175 1180
Val Glu Lys Thr Ile Lys Leu Arg Arg Ile Ser Asn Ser Asn Tyr Asn1185
1190 1195 1200Ser Asn Gln Met Ile
Ile Met Asp Ser Ile Gly Asp Asn Cys Thr Met 1205
1210 1215 Asn Phe Lys Thr Asn Asn Gly Asn Asp Ile
Gly Leu Leu Gly Phe His 1220 1225
1230 Leu Asn Asn Leu Val Ala Ser Ser Trp Tyr Tyr Lys Asn Ile Arg
Asn 1235 1240 1245 Asn
Thr Arg Asn Asn Gly Cys Phe Trp Ser Phe Ile Ser Lys Glu His 1250
1255 1260 Gly Trp Gln Glu1265
101251PRTClostridium butyricum 10Met Pro Thr Ile Asn Ser Phe Asn
Tyr Asn Asp Pro Val Asn Asn Arg1 5 10
15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Gln Phe
Tyr Lys Ser 20 25 30
Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile
35 40 45 Gly Thr Ile Pro
Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly 50 55
60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr
Leu Gln Ser Asp Gln Glu Lys65 70 75
80 Asp Lys Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile
Asn Asp 85 90 95
Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro
100 105 110 Tyr Leu Gly Asn Asp
Asn Thr Pro Asp Gly Asp Phe Ile Ile Asn Asp 115
120 125 Ala Ser Ala Val Pro Ile Gln Phe Ser
Asn Gly Ser Gln Ser Ile Leu 130 135
140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu
Phe Glu Thr145 150 155
160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His
165 170 175 Gly Phe Gly Ser
Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180
185 190 Arg Phe Lys Asp Asn Ser Met Asn Glu
Phe Ile Gln Asp Pro Ala Leu 195 200
205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr
Gly Ala 210 215 220
Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225
230 235 240 Ile Thr Asn Ile Arg
Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245
250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala
Gln Ser Asn Asp Ile Tyr 260 265
270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser
Lys 275 280 285 Val
Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290
295 300 Ala Lys Tyr Gly Leu Asp
Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310
315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu
Tyr Ser Phe Thr Glu 325 330
335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile
340 345 350 Gly Gln Tyr
Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355
360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn
Ile Asn Asn Leu Lys Val Asn Phe 370 375
380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr
Pro Ile Thr385 390 395
400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val
405 410 415 Ser Val Lys Gly
Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420
425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn
Ser Tyr Asn Asp Asp Asn Ile 435 440
445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn
Asn Tyr 450 455 460
Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465
470 475 480 Pro Gly Leu Ser Asp
Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485
490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr
Ser Asp Ile Glu Gln His 500 505
510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys
Val 515 520 525 Pro
Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530
535 540 Leu Leu Glu Gln Pro Lys
Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550
555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu
Phe Val Gly Trp Ile 565 570
575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr
580 585 590 Val Asp Lys
Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595
600 605 Ala Leu Asn Ile Gly Asn Glu Ala
Gln Lys Gly Asn Phe Lys Asp Ala 610 615
620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu
Pro Glu Leu625 630 635
640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser
645 650 655 Ser Asp Asn Lys
Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660
665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val
Tyr Ser Phe Ile Val Ser Asn 675 680
685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu
Gln Met 690 695 700
Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu705
710 715 720 Ser Lys Tyr Asn Ser
Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725
730 735 Lys Tyr Asp Ile Glu Gln Ile Glu Asn Glu
Leu Asn Gln Lys Val Ser 740 745
750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile
Ser 755 760 765 Tyr
Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770
775 780 Tyr Asp Glu Asn Val Lys
Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His785 790
795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu
Asn Ser Met Val Ile 805 810
815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp
820 825 830 Asp Lys Ile
Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835
840 845 Ser Ser Ser Val Leu Asn Met Arg
Tyr Lys Asn Asp Lys Tyr Val Asp 850 855
860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp
Val Tyr Lys865 870 875
880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser
885 890 895 Glu Val Asn Ile
Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900
905 910 Lys Asn Phe Ser Ile Ser Phe Trp Val
Arg Ile Pro Asn Tyr Asp Asn 915 920
925 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys
Met Arg 930 935 940
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile945
950 955 960 Trp Thr Leu Gln Asp
Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn 965
970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe 980 985
990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile
Asn 995 1000 1005 Gly
Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010
1015 1020 Val Ser Asp Asn Ile Leu
Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025 1030
1035 1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp
Lys Glu Leu Asp Glu 1045 1050
1055 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu
1060 1065 1070 Lys Asp Phe
Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075
1080 1085 Leu Asn Val Leu Lys Pro Asn Asn
Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095
1100 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu
Ala Asn Arg1105 1110 1115
1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser
1125 1130 1135 Thr Asn Asp Asn
Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140
1145 1150 Val Ala Ser Lys Thr His Leu Leu Pro
Leu Tyr Ala Asp Thr Ala Thr 1155 1160
1165 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn
Arg Phe 1170 1175 1180
Asn Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe1185
1190 1195 1200Lys Asn Asn Asn Gly
Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp 1205
1210 1215 Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr
His Met Arg Asp Asn Thr 1220 1225
1230 Asn Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly
Trp 1235 1240 1245 Gln
Glu Lys 1250 111035PRTBos taurus 11Met Gly Ser Lys Arg Ser Val Pro
Ser Arg His Arg Ser Leu Thr Thr1 5 10
15 Tyr Glu Val Met Phe Ala Val Leu Phe Val Ile Leu Val
Ala Leu Cys 20 25 30
Ala Gly Leu Ile Ala Val Ser Trp Leu Ser Ile Gln Gly Ser Val Lys
35 40 45 Asp Ala Ala Phe
Gly Lys Ser His Glu Ala Arg Gly Thr Leu Lys Ile 50 55
60 Ile Ser Gly Ala Thr Tyr Asn Pro His
Leu Gln Asp Lys Leu Ser Val65 70 75
80 Asp Phe Lys Val Leu Ala Phe Asp Ile Gln Gln Met Ile Asp
Asp Ile 85 90 95
Phe Gln Ser Ser Asn Leu Lys Asn Glu Tyr Lys Asn Ser Arg Val Leu
100 105 110 Gln Phe Glu Asn Gly
Ser Ile Ile Val Ile Phe Asp Leu Leu Phe Asp 115
120 125 Gln Trp Val Ser Asp Lys Asn Val Lys
Glu Glu Leu Ile Gln Gly Ile 130 135
140 Glu Ala Asn Lys Ser Ser Gln Leu Val Thr Phe His Ile
Asp Leu Asn145 150 155
160 Ser Ile Asp Ile Thr Ala Ser Leu Glu Asn Phe Ser Thr Ile Ser Pro
165 170 175 Ala Thr Thr Ser
Glu Lys Leu Thr Thr Ser Ile Pro Leu Ala Thr Pro 180
185 190 Gly Asn Val Ser Ile Glu Cys Pro Pro
Asp Ser Arg Leu Cys Ala Asp 195 200
205 Ala Leu Lys Cys Ile Ala Ile Asp Leu Phe Cys Asp Gly Glu
Leu Asn 210 215 220
Cys Pro Asp Gly Ser Asp Glu Asp Asn Lys Thr Cys Ala Thr Ala Cys225
230 235 240 Asp Gly Arg Phe Leu
Leu Thr Gly Ser Ser Gly Ser Phe Glu Ala Leu 245
250 255 His Tyr Pro Lys Pro Ser Asn Asn Thr Ser
Ala Val Cys Arg Trp Ile 260 265
270 Ile Arg Val Asn Gln Gly Leu Ser Ile Gln Leu Asn Phe Asp Tyr
Phe 275 280 285 Asn
Thr Tyr Tyr Ala Asp Val Leu Asn Ile Tyr Glu Gly Met Gly Ser 290
295 300 Ser Lys Ile Leu Arg Ala
Ser Leu Trp Ser Asn Asn Pro Gly Ile Ile305 310
315 320 Arg Ile Phe Ser Asn Gln Val Thr Ala Thr Phe
Leu Ile Gln Ser Asp 325 330
335 Glu Ser Asp Tyr Ile Gly Phe Lys Val Thr Tyr Thr Ala Phe Asn Ser
340 345 350 Lys Glu Leu
Asn Asn Tyr Glu Lys Ile Asn Cys Asn Phe Glu Asp Gly 355
360 365 Phe Cys Phe Trp Ile Gln Asp Leu
Asn Asp Asp Asn Glu Trp Glu Arg 370 375
380 Thr Gln Gly Ser Thr Phe Pro Pro Ser Thr Gly Pro Thr
Phe Asp His385 390 395
400 Thr Phe Gly Asn Glu Ser Gly Phe Tyr Ile Ser Thr Pro Thr Gly Pro
405 410 415 Gly Gly Arg Arg
Glu Arg Val Gly Leu Leu Thr Leu Pro Leu Asp Pro 420
425 430 Thr Pro Glu Gln Ala Cys Leu Ser Phe
Trp Tyr Tyr Met Tyr Gly Glu 435 440
445 Asn Val Tyr Lys Leu Ser Ile Asn Ile Ser Ser Asp Gln Asn
Met Glu 450 455 460
Lys Thr Ile Phe Gln Lys Glu Gly Asn Tyr Gly Gln Asn Trp Asn Tyr465
470 475 480 Gly Gln Val Thr Leu
Asn Glu Thr Val Glu Phe Lys Val Ser Phe Tyr 485
490 495 Gly Phe Lys Asn Gln Ile Leu Ser Asp Ile
Ala Leu Asp Asp Ile Ser 500 505
510 Leu Thr Tyr Gly Ile Cys Asn Val Ser Val Tyr Pro Glu Pro Thr
Leu 515 520 525 Val
Pro Thr Pro Pro Pro Glu Leu Pro Thr Asp Cys Gly Gly Pro His 530
535 540 Asp Leu Trp Glu Pro Asn
Thr Thr Phe Thr Ser Ile Asn Phe Pro Asn545 550
555 560 Ser Tyr Pro Asn Gln Ala Phe Cys Ile Trp Asn
Leu Asn Ala Gln Lys 565 570
575 Gly Lys Asn Ile Gln Leu His Phe Gln Glu Phe Asp Leu Glu Asn Ile
580 585 590 Ala Asp Val
Val Glu Ile Arg Asp Gly Glu Gly Asp Asp Ser Leu Phe 595
600 605 Leu Ala Val Tyr Thr Gly Pro Gly
Pro Val Asn Asp Val Phe Ser Thr 610 615
620 Thr Asn Arg Met Thr Val Leu Phe Ile Thr Asp Asn Met
Leu Ala Lys625 630 635
640 Gln Gly Phe Lys Ala Asn Phe Thr Thr Gly Tyr Gly Leu Gly Ile Pro
645 650 655 Glu Pro Cys Lys
Glu Asp Asn Phe Gln Cys Lys Asp Gly Glu Cys Ile 660
665 670 Pro Leu Val Asn Leu Cys Asp Gly Phe
Pro His Cys Lys Asp Gly Ser 675 680
685 Asp Glu Ala His Cys Val Arg Leu Phe Asn Gly Thr Thr Asp
Ser Ser 690 695 700
Gly Leu Val Gln Phe Arg Ile Gln Ser Ile Trp His Val Ala Cys Ala705
710 715 720 Glu Asn Trp Thr Thr
Gln Ile Ser Asp Asp Val Cys Gln Leu Leu Gly 725
730 735 Leu Gly Thr Gly Asn Ser Ser Val Pro Thr
Phe Ser Thr Gly Gly Gly 740 745
750 Pro Tyr Val Asn Leu Asn Thr Ala Pro Asn Gly Ser Leu Ile Leu
Thr 755 760 765 Pro
Ser Gln Gln Cys Leu Glu Asp Ser Leu Ile Leu Leu Gln Cys Asn 770
775 780 Tyr Lys Ser Cys Gly Lys
Lys Leu Val Thr Gln Glu Val Ser Pro Lys785 790
795 800 Ile Val Gly Gly Ser Asp Ser Arg Glu Gly Ala
Trp Pro Trp Val Val 805 810
815 Ala Leu Tyr Phe Asp Asp Gln Gln Val Cys Gly Ala Ser Leu Val Ser
820 825 830 Arg Asp Trp
Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Met 835
840 845 Glu Pro Ser Lys Trp Lys Ala Val
Leu Gly Leu His Met Ala Ser Asn 850 855
860 Leu Thr Ser Pro Gln Ile Glu Thr Arg Leu Ile Asp Gln
Ile Val Ile865 870 875
880 Asn Pro His Tyr Asn Lys Arg Arg Lys Asn Asn Asp Ile Ala Met Met
885 890 895 His Leu Glu Met
Lys Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys 900
905 910 Leu Pro Glu Glu Asn Gln Val Phe Pro
Pro Gly Arg Ile Cys Ser Ile 915 920
925 Ala Gly Trp Gly Ala Leu Ile Tyr Gln Gly Ser Thr Ala Asp
Val Leu 930 935 940
Gln Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Lys Cys Gln Gln Gln945
950 955 960 Met Pro Glu Tyr Asn
Ile Thr Glu Asn Met Val Cys Ala Gly Tyr Glu 965
970 975 Ala Gly Gly Val Asp Ser Cys Gln Gly Asp
Ser Gly Gly Pro Leu Met 980 985
990 Cys Gln Glu Asn Asn Arg Trp Leu Leu Ala Gly Val Thr Ser Phe
Gly 995 1000 1005 Tyr
Gln Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Pro 1010
1015 1020 Arg Phe Thr Glu Trp Ile
Gln Ser Phe Leu His1025 1030
103512183PRTHuman rhinovirus C 12Gly Pro Glu His Glu Phe Leu Asn Ala Leu
Ile Arg Arg Asn Cys His1 5 10
15 Ile Ile Thr Thr Asp Lys Gly Glu Phe Asn Leu Leu Gly Ile Tyr
Ser 20 25 30 Asn
Cys Ala Val Val Pro Thr His Ala Glu Pro Gly Asp Val Val Asp 35
40 45 Ile Asp Gly Arg Leu Val
Arg Val Leu Lys Gln Gln Val Leu Thr Asp 50 55
60 Met Asn Asp Val Asp Thr Glu Val Thr Val Leu
Trp Leu Asp Gln Asn65 70 75
80 Glu Lys Phe Arg Asp Ile Arg Arg Phe Ile Pro Glu His Gln Gln Asp
85 90 95 Trp His Asn
Ile His Leu Ala Thr Asn Val Thr Lys Phe Pro Met Leu 100
105 110 Asn Val Glu Val Gly His Thr Val
Pro Tyr Gly Glu Ile Asn Leu Ser 115 120
125 Gly Asn Ala Thr Cys Arg Leu Tyr Lys Tyr Asp Tyr Pro
Thr Gln Pro 130 135 140
Gly Gln Cys Gly Ala Val Leu Ala Asn Thr Gly Asn Ile Ile Gly Ile145
150 155 160 His Val Gly Gly Asn
Gly Arg Val Gly Tyr Ala Ala Ala Leu Leu Arg 165
170 175 Lys Tyr Phe Ala Glu Glu Gln
180 13183PRTHuman enterovirus 71 13Gly Pro Ser Leu Asp Phe
Ala Leu Ser Leu Leu Arg Arg Asn Val Arg1 5
10 15 Gln Val Gln Thr Asp Gln Gly His Phe Thr Met
Leu Gly Val Arg Asp 20 25 30
Arg Leu Ala Val Leu Pro Arg His Ser Gln Pro Gly Lys Thr Ile Trp
35 40 45 Ile Glu His
Lys Leu Val Asn Val Leu Asp Ala Val Glu Leu Val Asp 50
55 60 Glu Gln Gly Val Asn Leu Glu Leu
Thr Leu Ile Thr Leu Asp Thr Asn65 70 75
80 Glu Lys Phe Arg Asp Ile Thr Lys Phe Ile Pro Glu Asn
Ile Ser Thr 85 90 95
Ala Ser Asp Ala Thr Leu Val Ile Asn Thr Glu His Met Pro Ser Met
100 105 110 Phe Val Pro Val Gly
Asp Val Val Gln Tyr Gly Phe Leu Asn Leu Ser 115
120 125 Gly Lys Pro Thr His Arg Thr Met Met
Tyr Asn Phe Pro Thr Lys Ala 130 135
140 Gly Gln Cys Gly Gly Val Val Thr Ser Val Gly Lys Val
Ile Gly Ile145 150 155
160 His Ile Gly Gly Asn Gly Arg Gln Gly Phe Cys Ala Gly Leu Lys Arg
165 170 175 Ser Tyr Phe Ala
Ser Glu Gln 180 143054PRTPotyvirus 14Met Ala Leu
Ile Phe Gly Thr Val Asn Ala Asn Ile Leu Lys Glu Val1 5
10 15 Phe Gly Gly Ala Arg Met Ala Cys
Val Thr Ser Ala His Met Ala Gly 20 25
30 Ala Asn Gly Ser Ile Leu Lys Lys Ala Glu Glu Thr Ser
Arg Ala Ile 35 40 45
Met His Lys Pro Val Ile Phe Gly Glu Asp Tyr Ile Thr Glu Ala Asp 50
55 60 Leu Pro Tyr Thr Pro
Leu His Leu Glu Val Asp Ala Glu Met Glu Arg65 70
75 80 Met Tyr Tyr Leu Gly Arg Arg Ala Leu Thr
His Gly Lys Arg Arg Lys 85 90
95 Val Ser Val Asn Asn Lys Arg Asn Arg Arg Arg Lys Val Ala Lys
Thr 100 105 110 Tyr
Val Gly Arg Asp Ser Ile Val Glu Lys Ile Val Val Pro His Thr 115
120 125 Glu Arg Lys Val Asp Thr
Thr Ala Ala Val Glu Asp Ile Cys Asn Glu 130 135
140 Ala Thr Thr Gln Leu Val His Asn Ser Met Pro
Lys Arg Lys Lys Gln145 150 155
160 Lys Asn Phe Leu Pro Ala Thr Ser Leu Ser Asn Val Tyr Ala Gln Thr
165 170 175 Trp Ser Ile
Val Arg Lys Arg His Met Gln Val Glu Ile Ile Ser Lys 180
185 190 Lys Ser Val Arg Ala Arg Val Lys
Arg Phe Glu Gly Ser Val Gln Leu 195 200
205 Phe Ala Ser Val Arg His Met Tyr Gly Glu Arg Lys Arg
Val Asp Leu 210 215 220
Arg Ile Asp Asn Trp Gln Gln Glu Thr Leu Leu Asp Leu Ala Lys Arg225
230 235 240 Phe Lys Asn Glu Arg
Val Asp Gln Ser Lys Leu Thr Phe Gly Ser Ser 245
250 255 Gly Leu Val Leu Arg Gln Gly Ser Tyr Gly
Pro Ala His Trp Tyr Arg 260 265
270 His Gly Met Phe Ile Val Arg Gly Arg Ser Asp Gly Met Leu Val
Asp 275 280 285 Ala
Arg Ala Lys Val Thr Phe Ala Val Cys His Ser Met Thr His Tyr 290
295 300 Ser Asp Lys Ser Ile Ser
Glu Ala Phe Phe Ile Pro Tyr Ser Lys Lys305 310
315 320 Phe Leu Glu Leu Arg Pro Asp Gly Ile Ser His
Glu Cys Thr Arg Gly 325 330
335 Val Ser Val Glu Arg Cys Gly Glu Val Ala Ala Ile Leu Thr Gln Ala
340 345 350 Leu Ser Pro
Cys Gly Lys Ile Thr Cys Lys Arg Cys Met Val Glu Thr 355
360 365 Pro Asp Ile Val Glu Gly Glu Ser
Gly Glu Ser Val Thr Asn Gln Gly 370 375
380 Lys Leu Leu Ala Met Leu Lys Glu Gln Tyr Pro Asp Phe
Pro Met Ala385 390 395
400 Glu Lys Leu Leu Thr Arg Phe Leu Gln Gln Lys Ser Leu Val Asn Thr
405 410 415 Asn Leu Thr Ala
Cys Val Ser Val Lys Gln Leu Ile Gly Asp Arg Lys 420
425 430 Gln Ala Pro Phe Thr His Val Leu Ala
Val Ser Glu Ile Leu Phe Lys 435 440
445 Gly Asn Lys Leu Thr Gly Ala Asp Leu Glu Glu Ala Ser Thr
His Met 450 455 460
Leu Glu Ile Ala Arg Phe Leu Asn Asn Arg Thr Glu Asn Met Arg Ile465
470 475 480 Gly His Leu Gly Ser
Phe Arg Asn Lys Ile Ser Ser Lys Ala His Val 485
490 495 Asn Asn Ala Leu Met Cys Asp Asn Gln Leu
Asp Gln Asn Gly Asn Phe 500 505
510 Ile Trp Gly Leu Arg Gly Ala His Ala Lys Arg Phe Leu Lys Gly
Phe 515 520 525 Phe
Thr Glu Ile Asp Pro Asn Glu Gly Tyr Asp Lys Tyr Val Ile Arg 530
535 540 Lys His Ile Arg Gly Ser
Arg Lys Leu Ala Ile Gly Asn Leu Ile Met545 550
555 560 Ser Thr Asp Phe Gln Thr Leu Arg Gln Gln Ile
Gln Gly Glu Thr Ile 565 570
575 Glu Arg Lys Glu Ile Gly Asn His Cys Ile Ser Met Arg Asn Gly Asn
580 585 590 Tyr Val Tyr
Pro Cys Cys Cys Val Thr Leu Glu Asp Gly Lys Ala Gln 595
600 605 Tyr Ser Asp Leu Lys His Pro Thr
Lys Arg His Leu Val Ile Gly Asn 610 615
620 Ser Gly Asp Ser Lys Tyr Leu Asp Leu Pro Val Leu Asn
Glu Glu Lys625 630 635
640 Met Tyr Ile Ala Asn Glu Gly Tyr Cys Tyr Met Asn Ile Phe Phe Ala
645 650 655 Leu Leu Val Asn
Val Lys Glu Glu Asp Ala Lys Asp Phe Thr Lys Phe 660
665 670 Ile Arg Asp Thr Ile Val Pro Lys Leu
Gly Ala Trp Pro Thr Met Gln 675 680
685 Asp Val Ala Thr Ala Cys Tyr Leu Leu Ser Ile Leu Tyr Pro
Asp Val 690 695 700
Leu Arg Ala Glu Leu Pro Arg Ile Leu Val Asp His Asp Asn Lys Thr705
710 715 720 Met His Val Leu Asp
Ser Tyr Gly Ser Arg Thr Thr Gly Tyr His Met 725
730 735 Leu Lys Met Asn Thr Thr Ser Gln Leu Ile
Glu Phe Val His Ser Gly 740 745
750 Leu Glu Ser Glu Met Lys Thr Tyr Asn Val Gly Gly Met Asn Arg
Asp 755 760 765 Val
Val Thr Gln Gly Ala Ile Glu Met Leu Ile Lys Ser Ile Tyr Lys 770
775 780 Pro His Leu Met Lys Gln
Leu Leu Glu Glu Glu Pro Tyr Ile Ile Val785 790
795 800 Leu Ala Ile Val Ser Pro Ser Ile Leu Ile Ala
Met Tyr Asn Ser Gly 805 810
815 Thr Phe Glu Gln Ala Leu Gln Met Trp Leu Pro Asn Thr Met Arg Leu
820 825 830 Ala Asn Leu
Ala Ala Ile Leu Ser Ala Leu Ala Gln Lys Leu Thr Leu 835
840 845 Ala Asp Leu Phe Val Gln Gln Arg
Asn Leu Ile Asn Glu Tyr Ala Gln 850 855
860 Val Ile Leu Asp Asn Leu Ile Asp Gly Val Arg Val Asn
His Ser Leu865 870 875
880 Ser Leu Ala Met Glu Ile Val Thr Ile Lys Leu Ala Thr Gln Glu Met
885 890 895 Asp Met Ala Leu
Arg Glu Gly Gly Tyr Ala Val Thr Ser Glu Lys Val 900
905 910 His Glu Met Leu Glu Lys Asn Tyr Val
Lys Ala Leu Lys Asp Ala Trp 915 920
925 Asp Glu Leu Thr Trp Leu Glu Lys Phe Ser Ala Ile Arg His
Ser Arg 930 935 940
Lys Leu Leu Lys Phe Gly Arg Lys Pro Leu Ile Met Lys Asn Thr Val945
950 955 960 Asp Cys Gly Gly His
Ile Asp Leu Ser Val Lys Ser Leu Phe Lys Phe 965
970 975 His Leu Glu Leu Leu Lys Gly Thr Ile Ser
Arg Ala Val Asn Gly Gly 980 985
990 Ala Arg Lys Val Arg Val Ala Lys Asn Ala Met Thr Lys Gly Val
Phe 995 1000 1005 Leu
Lys Ile Tyr Ser Met Leu Pro Asp Val Tyr Lys Phe Ile Thr Val 1010
1015 1020 Ser Ser Val Leu Ser Leu
Leu Leu Thr Phe Leu Phe Gln Ile Asp Cys1025 1030
1035 1040Met Ile Arg Ala His Arg Glu Ala Lys Val Ala
Ala Gln Leu Gln Lys 1045 1050
1055 Glu Ser Glu Trp Asp Asn Ile Ile Asn Arg Thr Phe Gln Tyr Ser Lys
1060 1065 1070 Leu Glu Asn
Pro Ile Gly Tyr Arg Ser Thr Ala Glu Glu Arg Leu Gln 1075
1080 1085 Ser Glu His Pro Glu Ala Phe Glu
Tyr Tyr Lys Phe Cys Ile Gly Lys 1090 1095
1100 Glu Asp Leu Val Glu Gln Ala Lys Gln Pro Glu Ile Ala
Tyr Phe Glu1105 1110 1115
1120Lys Ile Ile Ala Phe Ile Thr Leu Val Leu Met Ala Phe Asp Ala Glu
1125 1130 1135 Arg Ser Asp Gly
Val Phe Lys Ile Leu Asn Lys Phe Lys Gly Ile Leu 1140
1145 1150 Ser Ser Thr Glu Arg Glu Ile Ile Tyr
Thr Gln Ser Leu Asp Asp Tyr 1155 1160
1165 Val Thr Thr Phe Asp Asp Asn Met Thr Ile Asn Leu Glu Leu
Asn Met 1170 1175 1180
Asp Glu Leu His Lys Thr Ser Leu Pro Gly Val Thr Phe Lys Gln Trp1185
1190 1195 1200Trp Asn Asn Gln Ile
Ser Arg Gly Asn Val Lys Pro His Tyr Arg Thr 1205
1210 1215 Glu Gly His Phe Met Glu Phe Thr Arg Asp
Thr Ala Ala Ser Val Ala 1220 1225
1230 Ser Glu Ile Ser His Ser Pro Ala Arg Asp Phe Leu Val Arg Gly
Ala 1235 1240 1245 Val
Gly Ser Gly Lys Ser Thr Gly Leu Pro Tyr His Leu Ser Lys Arg 1250
1255 1260 Gly Arg Val Leu Met Leu
Glu Pro Thr Arg Pro Leu Thr Asp Asn Met1265 1270
1275 1280His Lys Gln Leu Arg Ser Glu Pro Phe Asn Cys
Phe Pro Thr Leu Arg 1285 1290
1295 Met Arg Gly Lys Ser Thr Phe Gly Ser Ser Pro Ile Thr Val Met Thr
1300 1305 1310 Ser Gly Phe
Ala Leu His His Phe Ala Arg Asn Ile Ala Glu Val Lys 1315
1320 1325 Thr Tyr Asp Phe Val Ile Ile Asp
Glu Cys His Val Asn Asp Ala Ser 1330 1335
1340 Ala Ile Ala Phe Arg Asn Leu Leu Phe Glu His Glu Phe
Glu Gly Lys1345 1350 1355
1360Val Leu Lys Val Ser Ala Thr Pro Pro Gly Arg Glu Val Glu Phe Thr
1365 1370 1375 Thr Gln Phe Pro
Val Lys Leu Lys Ile Glu Glu Ala Leu Ser Phe Gln 1380
1385 1390 Glu Phe Val Ser Leu Gln Gly Thr Gly
Ala Asn Ala Asp Val Ile Ser 1395 1400
1405 Cys Gly Asp Asn Ile Leu Val Tyr Val Ala Ser Tyr Asn Asp
Val Asp 1410 1415 1420
Ser Leu Gly Lys Leu Leu Val Gln Lys Gly Tyr Lys Val Ser Lys Ile1425
1430 1435 1440Asp Gly Arg Thr Met
Lys Ser Gly Gly Thr Glu Ile Ile Thr Glu Gly 1445
1450 1455 Thr Ser Val Lys Lys His Phe Ile Val Ala
Thr Asn Ile Ile Glu Asn 1460 1465
1470 Gly Val Thr Ile Asp Ile Asp Val Val Val Asp Phe Gly Thr Lys
Val 1475 1480 1485 Val
Pro Val Leu Asp Val Asp Asn Arg Ala Val Gln Tyr Asn Lys Thr 1490
1495 1500 Val Val Ser Tyr Gly Glu
Arg Ile Gln Lys Leu Gly Arg Val Gly Arg1505 1510
1515 1520His Lys Glu Gly Val Ala Leu Arg Ile Gly Gln
Thr Asn Lys Thr Leu 1525 1530
1535 Val Glu Ile Pro Glu Met Val Ala Thr Glu Ala Ala Phe Leu Cys Phe
1540 1545 1550 Met Tyr Asn
Leu Pro Val Thr Thr Gln Ser Val Ser Thr Thr Leu Leu 1555
1560 1565 Glu Asn Ala Thr Leu Leu Gln Ala
Arg Thr Met Ala Gln Phe Glu Leu 1570 1575
1580 Ser Tyr Phe Tyr Thr Ile Asn Phe Val Arg Phe Asp Gly
Ser Met His1585 1590 1595
1600Pro Val Ile His Asp Lys Leu Lys Arg Phe Lys Leu His Thr Cys Glu
1605 1610 1615 Thr Phe Leu Asn
Lys Leu Ala Ile Pro Asn Lys Gly Leu Ser Ser Trp 1620
1625 1630 Leu Thr Ser Gly Glu Tyr Lys Arg Leu
Gly Tyr Ile Ala Glu Asp Ala 1635 1640
1645 Gly Ile Arg Ile Pro Phe Val Cys Lys Glu Ile Pro Asp Ser
Leu His 1650 1655 1660
Glu Glu Ile Trp His Ile Val Val Ala His Lys Gly Asp Ser Gly Ile1665
1670 1675 1680Gly Arg Leu Thr Ser
Val Gln Ala Ala Lys Val Val Tyr Thr Leu Gln 1685
1690 1695 Thr Asp Val His Ser Ile Ala Arg Thr Leu
Ala Cys Ile Asn Arg Arg 1700 1705
1710 Ile Ala Asp Glu Gln Met Lys Gln Ser His Phe Glu Ala Ala Thr
Gly 1715 1720 1725 Arg
Ala Phe Ser Phe Thr Asn Tyr Ser Ile Gln Ser Ile Phe Asp Thr 1730
1735 1740 Leu Lys Ala Asn Tyr Ala
Thr Lys His Thr Lys Glu Asn Ile Ala Val1745 1750
1755 1760Leu Gln Gln Ala Lys Asp Gln Leu Leu Glu Phe
Ser Asn Leu Ala Lys 1765 1770
1775 Asp Gln Asp Val Thr Gly Ile Ile Gln Asp Phe Asn His Leu Glu Thr
1780 1785 1790 Ile Tyr Leu
Gln Ser Asp Ser Glu Val Ala Lys His Leu Lys Leu Lys 1795
1800 1805 Ser His Trp Asn Lys Ser Gln Ile
Thr Arg Asp Ile Ile Ile Ala Leu 1810 1815
1820 Ser Val Leu Ile Gly Gly Gly Trp Met Leu Ala Thr Tyr
Phe Lys Asp1825 1830 1835
1840Lys Phe Asn Glu Pro Val Tyr Phe Gln Gly Lys Lys Asn Gln Lys His
1845 1850 1855 Lys Leu Lys Met
Arg Glu Ala Arg Gly Ala Arg Gly Gln Tyr Glu Val 1860
1865 1870 Ala Ala Glu Pro Glu Ala Leu Glu His
Tyr Phe Gly Ser Ala Tyr Asn 1875 1880
1885 Asn Lys Gly Lys Arg Lys Gly Thr Thr Arg Gly Met Gly Ala
Lys Ser 1890 1895 1900
Arg Lys Phe Ile Asn Met Tyr Gly Phe Asp Pro Thr Asp Phe Ser Tyr1905
1910 1915 1920Ile Arg Phe Val Asp
Pro Leu Thr Gly His Thr Ile Asp Glu Ser Thr 1925
1930 1935 Asn Ala Pro Ile Asp Leu Val Gln His Glu
Phe Gly Lys Val Arg Thr 1940 1945
1950 Arg Met Leu Ile Asp Asp Glu Ile Glu Pro Gln Ser Leu Ser Thr
His 1955 1960 1965 Thr
Thr Ile His Ala Tyr Leu Val Asn Ser Gly Thr Lys Lys Val Leu 1970
1975 1980 Lys Val Asp Leu Thr Pro
His Ser Ser Leu Arg Ala Ser Glu Lys Ser1985 1990
1995 2000Thr Ala Ile Met Gly Phe Pro Glu Arg Glu Asn
Glu Leu Arg Gln Thr 2005 2010
2015 Gly Met Ala Val Pro Val Ala Tyr Asp Gln Leu Pro Pro Lys Asn Glu
2020 2025 2030 Asp Leu Thr
Phe Glu Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr 2035
2040 2045 Asn Pro Ile Ser Ser Thr Ile Cys
His Leu Thr Asn Glu Ser Asp Gly 2050 2055
2060 His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe
Ile Ile Thr2065 2070 2075
2080Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser
2085 2090 2095 Leu His Gly Val
Phe Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His 2100
2105 2110 Leu Ile Asp Gly Arg Asp Met Ile Ile
Ile Arg Met Pro Lys Asp Phe 2115 2120
2125 Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg
Glu Glu 2130 2135 2140
Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser2145
2150 2155 2160Met Val Ser Asp Thr
Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe 2165
2170 2175 Trp Lys His Trp Ile Gln Thr Lys Asp Gly
Gln Cys Gly Ser Pro Leu 2180 2185
2190 Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser
Asn 2195 2200 2205 Phe
Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met 2210
2215 2220 Glu Leu Leu Thr Asn Gln
Glu Ala Gln Gln Trp Val Ser Gly Trp Arg2225 2230
2235 2240Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His
Lys Val Phe Met Ser 2245 2250
2255 Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met
2260 2265 2270 Asn Glu Leu
Val Tyr Ser Gln Gly Glu Lys Arg Lys Trp Val Val Glu 2275
2280 2285 Ala Leu Ser Gly Asn Leu Arg Pro
Val Ala Glu Cys Pro Ser Gln Leu 2290 2295
2300 Val Thr Lys His Val Val Lys Gly Lys Cys Pro Leu Phe
Glu Leu Tyr2305 2310 2315
2320Leu Gln Leu Asn Pro Glu Lys Glu Ala Tyr Phe Lys Pro Met Met Gly
2325 2330 2335 Ala Tyr Lys Pro
Ser Arg Leu Asn Arg Glu Ala Phe Leu Lys Asp Ile 2340
2345 2350 Leu Lys Tyr Ala Ser Glu Ile Glu Ile
Gly Asn Val Asp Cys Asp Leu 2355 2360
2365 Leu Glu Leu Ala Ile Ser Met Leu Val Thr Lys Leu Lys Ala
Leu Gly 2370 2375 2380
Phe Pro Thr Val Asn Tyr Ile Thr Asp Pro Glu Glu Ile Phe Ser Ala2385
2390 2395 2400Leu Asn Met Lys Ala
Ala Met Gly Ala Leu Tyr Lys Gly Lys Lys Lys 2405
2410 2415 Glu Ala Leu Ser Glu Leu Thr Leu Asp Glu
Gln Glu Ala Met Leu Lys 2420 2425
2430 Ala Ser Cys Leu Arg Leu Tyr Thr Gly Lys Leu Gly Ile Trp Asn
Gly 2435 2440 2445 Ser
Leu Lys Ala Glu Leu Arg Pro Ile Glu Lys Val Glu Asn Asn Lys 2450
2455 2460 Thr Arg Thr Phe Thr Ala
Ala Pro Ile Asp Thr Leu Leu Ala Gly Lys2465 2470
2475 2480Val Cys Val Asp Asp Phe Asn Asn Gln Phe Tyr
Asp Leu Asn Ile Lys 2485 2490
2495 Ala Pro Trp Thr Val Gly Met Thr Lys Phe Tyr Gln Gly Trp Asn Glu
2500 2505 2510 Leu Met Glu
Ala Leu Pro Ser Gly Trp Val Tyr Cys Asp Ala Asp Gly 2515
2520 2525 Ser Gln Phe Asp Ser Ser Leu Thr
Pro Phe Leu Ile Asn Ala Val Leu 2530 2535
2540 Lys Val Arg Leu Ala Phe Met Glu Glu Trp Asp Ile Gly
Glu Gln Met2545 2550 2555
2560Leu Arg Asn Leu Tyr Thr Glu Ile Val Tyr Thr Pro Ile Leu Thr Pro
2565 2570 2575 Asp Gly Thr Ile
Ile Lys Lys His Lys Gly Asn Asn Ser Gly Gln Pro 2580
2585 2590 Ser Thr Val Val Asp Asn Thr Leu Met
Val Ile Ile Ala Met Leu Tyr 2595 2600
2605 Thr Cys Glu Lys Cys Gly Ile Asn Lys Glu Glu Ile Val Tyr
Tyr Val 2610 2615 2620
Asn Gly Asp Asp Leu Leu Ile Ala Ile His Pro Asp Lys Ala Glu Arg2625
2630 2635 2640Leu Ser Arg Phe Lys
Glu Ser Phe Gly Glu Leu Gly Leu Lys Tyr Glu 2645
2650 2655 Phe Asp Cys Thr Thr Arg Asp Lys Thr Gln
Leu Trp Phe Met Ser His 2660 2665
2670 Arg Ala Leu Glu Arg Asp Gly Met Tyr Ile Pro Lys Leu Glu Glu
Glu 2675 2680 2685 Arg
Ile Val Ser Ile Leu Glu Trp Asp Arg Ser Lys Glu Pro Ser His 2690
2695 2700 Arg Leu Glu Ala Ile Cys
Ala Ser Met Ile Glu Ala Trp Gly Tyr Asp2705 2710
2715 2720Lys Leu Val Glu Glu Ile Arg Asn Phe Tyr Ala
Trp Val Leu Glu Gln 2725 2730
2735 Ala Pro Tyr Ser Gln Leu Ala Glu Glu Gly Lys Ala Pro Tyr Leu Ala
2740 2745 2750 Glu Thr Ala
Leu Lys Phe Leu Tyr Thr Ser Gln His Gly Thr Asn Ser 2755
2760 2765 Glu Ile Glu Glu Tyr Leu Lys Val
Leu Tyr Asp Tyr Asp Ile Pro Thr 2770 2775
2780 Thr Glu Asn Leu Tyr Phe Gln Ser Gly Thr Val Asp Ala
Gly Ala Asp2785 2790 2795
2800Ala Gly Lys Lys Lys Asp Gln Lys Asp Asp Lys Val Ala Glu Gln Ala
2805 2810 2815 Ser Lys Asp Arg
Asp Val Asn Ala Gly Thr Ser Gly Thr Phe Ser Val 2820
2825 2830 Pro Arg Ile Asn Ala Met Ala Thr Lys
Leu Gln Tyr Pro Arg Met Arg 2835 2840
2845 Gly Glu Val Val Val Asn Leu Asn His Leu Leu Gly Tyr Lys
Pro Gln 2850 2855 2860
Gln Ile Asp Leu Ser Asn Ala Arg Ala Thr His Glu Gln Phe Ala Ala2865
2870 2875 2880Trp His Gln Ala Val
Met Thr Ala Tyr Gly Val Asn Glu Glu Gln Met 2885
2890 2895 Lys Ile Leu Leu Asn Gly Phe Met Val Trp
Cys Ile Glu Asn Gly Thr 2900 2905
2910 Ser Pro Asn Leu Asn Gly Thr Trp Val Met Met Asp Gly Glu Asp
Gln 2915 2920 2925 Val
Ser Tyr Pro Leu Lys Pro Met Val Glu Asn Ala Gln Pro Thr Leu 2930
2935 2940 Arg Gln Ile Met Thr His
Phe Ser Asp Leu Ala Glu Ala Tyr Ile Glu2945 2950
2955 2960Met Arg Asn Arg Glu Arg Pro Tyr Met Pro Arg
Tyr Gly Leu Gln Arg 2965 2970
2975 Asn Ile Thr Asp Met Ser Leu Ser Arg Tyr Ala Phe Asp Phe Tyr Glu
2980 2985 2990 Leu Thr Ser
Lys Thr Pro Val Arg Ala Arg Glu Ala His Met Gln Met 2995
3000 3005 Lys Ala Ala Ala Val Arg Asn Ser
Gly Thr Arg Leu Phe Gly Leu Asp 3010 3015
3020 Gly Asn Val Gly Thr Ala Glu Glu Asp Thr Glu Arg His
Thr Ala His3025 3030 3035
3040Asp Val Asn Arg Asn Met His Thr Leu Leu Gly Val Arg Gln
3045 3050 15242PRTPotyvirus 15Gly Glu Ser
Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser1 5
10 15 Thr Ile Cys His Leu Thr Asn Glu
Ser Asp Gly His Thr Thr Ser Leu 20 25
30 Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys
His Leu Phe 35 40 45
Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe 50
55 60 Lys Val Lys Asn Thr
Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg65 70
75 80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp
Phe Pro Pro Phe Pro Gln 85 90
95 Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu
Val 100 105 110 Thr
Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser
Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu
Val Ser Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe
Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser
Gly Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Ser Lys Pro
Glu Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln16242PRTPotyvirusVARIANT216S219N 16Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Leu Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asn Thr Thr Thr Leu Gln Gln
His Leu Ile Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Ser Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln17242PRTPotyvirusVARIANT56L56V 17Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Val Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asn Thr Thr Thr Leu Gln Gln
His Leu Ile Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Gly Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln18242PRTPotyvirusVARIANT17T17V 18Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Leu Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asp Thr Thr Thr Leu Gln Gln
His Leu Val Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Ser Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln19242PRTPotyvirusVARIANT44N44V 19Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Val Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Val Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asn Thr Thr Thr Leu Gln Gln
His Leu Ile Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Gly Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln20242PRTPotyvirusVARIANT56L56V 20Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Val Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asp Thr Thr Thr Leu Gln Gln
His Leu Ile Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Gly Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln21242PRTPotyvirusVARIANT17T17S 21Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Val Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asp Thr Thr Thr Leu Gln Gln
His Leu Val Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Ser Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln22242PRTPotyvirusVARIANT17T17S 22Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Leu Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asp Thr Thr Thr Leu Gln Gln
His Leu Val Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Gly Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln23242PRTPotyvirusVARIANT17T17S 23Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser Ser1 5 10
15 Ser Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser Leu 20 25 30
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Val Lys His Leu Phe 35
40 45 Arg Arg Asn Asn Gly
Thr Leu Val Val Gln Ser Leu His Gly Val Phe 50 55
60 Lys Val Lys Asp Thr Thr Thr Leu Gln Gln
His Leu Val Asp Gly Arg65 70 75
80 Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
Gln 85 90 95 Lys
Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val
100 105 110 Thr Thr Asn Phe Gln
Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115
120 125 Ser Cys Thr Phe Pro Ser Gly Asp Gly
Ile Phe Trp Lys His Trp Ile 130 135
140 Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser
Thr Arg Asp145 150 155
160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn
165 170 175 Asn Tyr Phe Thr
Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 180
185 190 Gln Glu Ala Gln Gln Trp Val Ser Gly
Trp Arg Leu Asn Ala Asp Ser 195 200
205 Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu
Glu Pro 210 215 220
Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr225
230 235 240 Ser
Gln243023PRTPotyvirus 24Met Ala Ala Thr Met Ile Phe Gly Ser Phe Thr His
Asp Leu Leu Gly1 5 10 15
Lys Ala Met Ser Thr Ile His Ser Ala Val Thr Ala Glu Lys Asp Ile
20 25 30 Phe Ser Ser Ile
Lys Glu Arg Leu Glu Arg Lys Arg His Gly Lys Ile 35
40 45 Cys Arg Met Lys Asn Gly Ser Ile Tyr
Ile Lys Ala Ala Ser Ser Thr 50 55 60
Lys Val Glu Lys Ile Asn Ala Ala Ala Lys Lys Leu Ala Asp
Asp Lys65 70 75 80
Ala Ala Phe Leu Lys Ala Gln Pro Thr Ile Val Asp Lys Ile Ile Val
85 90 95 Asn Glu Lys Ile Gln
Val Val Glu Ala Glu Glu Val His Lys Arg Glu 100
105 110 Asp Val Gln Thr Val Phe Phe Lys Lys Thr
Lys Lys Arg Ala Pro Lys 115 120
125 Leu Arg Ala Thr Cys Ser Ser Ser Gly Leu Asp Asn Leu Tyr
Asn Ala 130 135 140
Val Ala Asn Ile Ala Lys Ala Ser Ser Leu Arg Val Glu Val Ile His145
150 155 160 Lys Lys Arg Val Cys
Gly Glu Phe Lys Gln Thr Arg Phe Gly Arg Ala 165
170 175 Leu Phe Ile Asp Val Ala His Ala Lys Gly
His Arg Arg Arg Ile Asp 180 185
190 Cys Arg Met His Arg Arg Glu Gln Arg Thr Met His Met Phe Met
Arg 195 200 205 Lys
Thr Thr Lys Thr Glu Val Arg Ser Lys His Leu Arg Lys Gly Asp 210
215 220 Ser Gly Ile Val Leu Leu
Thr Gln Lys Ile Lys Gly His Leu Ser Gly225 230
235 240 Val Arg Asp Glu Phe Phe Ile Val Arg Gly Thr
Cys Asp Asp Ser Leu 245 250
255 Leu Glu Ala Arg Ala Arg Phe Ser Gln Ser Ile Thr Leu Arg Ala Thr
260 265 270 His Phe Ser
Thr Gly Asp Ile Phe Trp Lys Gly Phe Asn Ala Ser Phe 275
280 285 Gln Glu Gln Lys Ala Ile Gly Leu
Asp His Thr Cys Thr Ser Asp Leu 290 295
300 Pro Val Glu Ala Cys Gly His Val Ala Ala Leu Met Cys
Gln Ser Leu305 310 315
320 Phe Pro Cys Gly Lys Ile Thr Cys Lys Arg Cys Ile Ala Asn Leu Ser
325 330 335 Asn Leu Asp Phe
Asp Thr Phe Ser Glu Leu Gln Gly Asp Arg Ala Met 340
345 350 Arg Ile Leu Asp Val Met Arg Ala Arg
Phe Pro Ser Phe Thr His Thr 355 360
365 Ile Arg Phe Leu His Asp Leu Phe Thr Gln Arg Arg Val Thr
Asn Pro 370 375 380
Asn Thr Ala Ala Phe Arg Glu Ile Leu Arg Leu Ile Gly Asp Arg Asn385
390 395 400 Glu Ala Pro Phe Ala
His Val Asn Arg Leu Asn Glu Ile Leu Leu Leu 405
410 415 Gly Ser Lys Ala Asn Pro Asp Ser Leu Ala
Lys Ala Ser Asp Ser Leu 420 425
430 Leu Glu Leu Ala Arg Tyr Leu Asn Asn Arg Thr Glu Asn Ile Arg
Asn 435 440 445 Gly
Ser Leu Lys His Phe Arg Asn Lys Ile Ser Ser Lys Ala His Ser 450
455 460 Asn Leu Ala Leu Ser Cys
Asp Asn Gln Leu Asp Gln Asn Gly Asn Phe465 470
475 480 Leu Trp Gly Leu Ala Gly Ile Ala Ala Lys Arg
Phe Leu Asn Asn Tyr 485 490
495 Phe Glu Thr Ile Asp Pro Glu Gln Gly Tyr Asp Lys Tyr Val Ile Arg
500 505 510 Lys Asn Pro
Asn Gly Glu Arg Lys Leu Ala Ile Gly Asn Phe Ile Ile 515
520 525 Ser Thr Asn Leu Glu Lys Leu Arg
Asp Gln Leu Glu Gly Glu Ser Ile 530 535
540 Ala Arg Val Gly Ile Thr Glu Glu Cys Val Ser Arg Lys
Asp Gly Asn545 550 555
560 Tyr Arg Tyr Pro Cys Cys Cys Val Thr Leu Glu Asp Gly Ser Pro Met
565 570 575 Tyr Ser Glu Leu
Lys Met Pro Thr Lys Asn His Leu Val Ile Gly Asn 580
585 590 Ser Gly Asp Pro Lys Tyr Leu Asp Leu
Pro Gly Glu Ile Ser Asn Leu 595 600
605 Met Tyr Ile Ala Lys Glu Gly Tyr Cys Tyr Ile Asn Ile Phe
Leu Ala 610 615 620
Met Leu Val Asn Val Asp Glu Ala Asn Ala Lys Asp Phe Thr Lys Arg625
630 635 640 Val Arg Asp Glu Ser
Val Gln Lys Leu Gly Lys Trp Pro Ser Leu Ile 645
650 655 Asp Val Ala Thr Glu Cys Ala Leu Leu Ser
Thr Tyr Tyr Pro Ala Ala 660 665
670 Ala Ser Ala Glu Leu Pro Arg Leu Leu Val Asp His Ala Gln Lys
Thr 675 680 685 Ile
His Val Val Asp Ser Tyr Gly Ser Leu Asn Thr Gly Tyr His Ile 690
695 700 Leu Lys Ala Asn Thr Val
Ser Gln Leu Glu Lys Phe Ala Ser Asn Thr705 710
715 720 Leu Glu Ser Pro Met Ala Gln Tyr Lys Val Gly
Gly Leu Val Tyr Ser 725 730
735 Glu Asn Asn Asp Ala Ser Ala Val Lys Ala Leu Thr Gln Ala Ile Phe
740 745 750 Arg Pro Asp
Val Leu Ser Glu Leu Ile Glu Lys Glu Pro Tyr Leu Met 755
760 765 Val Phe Ala Leu Val Ser Pro Gly
Ile Leu Met Ala Met Ser Asn Ser 770 775
780 Gly Ala Leu Glu Phe Gly Ile Ser Lys Trp Ile Ser Ser
Asp His Ser785 790 795
800 Leu Val Arg Met Ala Ser Ile Leu Lys Thr Leu Ala Ser Lys Val Ser
805 810 815 Val Ala Asp Thr
Leu Ala Leu Gln Lys His Ile Met Arg Gln Asn Ala 820
825 830 Asn Phe Leu Cys Gly Glu Leu Ile Asn
Gly Phe Gln Lys Lys Lys Ser 835 840
845 Tyr Thr His Ala Thr Arg Phe Leu Leu Met Ile Ser Glu Glu
Asn Glu 850 855 860
Met Asp Asp Pro Val Leu Asn Ala Gly Tyr Arg Val Leu Glu Ala Ser865
870 875 880 Ser His Glu Ile Met
Glu Lys Thr Tyr Leu Ala Leu Leu Glu Thr Ser 885
890 895 Trp Ser Asp Leu Ser Leu Tyr Gly Lys Phe
Lys Ser Ile Trp Phe Thr 900 905
910 Arg Lys His Phe Gly Arg Tyr Lys Ala Glu Leu Phe Pro Lys Glu
Gln 915 920 925 Thr
Asp Leu Gln Gly Arg Tyr Ser Asn Ser Leu Arg Phe His Tyr Gln 930
935 940 Ser Thr Leu Lys Arg Leu
Arg Asn Lys Gly Ser Leu Cys Arg Glu Arg945 950
955 960 Phe Leu Glu Ser Ile Ser Ser Ala Arg Arg Arg
Thr Thr Cys Ala Val 965 970
975 Phe Ser Leu Leu His Lys Ala Phe Pro Asp Val Leu Lys Phe Ile Asn
980 985 990 Thr Leu Val
Ile Val Ser Leu Ser Met Gln Ile Tyr Tyr Met Leu Val 995
1000 1005 Ala Ile Ile His Glu His Arg Ala
Ala Lys Ile Lys Ser Ala Gln Leu 1010 1015
1020 Glu Glu Arg Val Leu Glu Asp Lys Thr Met Leu Leu Tyr
Asp Asp Phe1025 1030 1035
1040Lys Ala Lys Leu Pro Glu Gly Ser Phe Glu Glu Phe Leu Glu Tyr Thr
1045 1050 1055 Arg Gln Arg Asp
Lys Glu Val Tyr Glu Tyr Leu Met Met Glu Thr Thr 1060
1065 1070 Glu Ile Val Glu Phe Gln Ala Lys Asn
Thr Gly Gln Ala Ser Leu Glu 1075 1080
1085 Arg Ile Ile Ala Phe Val Ser Leu Thr Leu Met Leu Phe Asp
Asn Glu 1090 1095 1100
Arg Ser Asp Cys Val Tyr Lys Ile Leu Thr Lys Phe Lys Gly Ile Leu1105
1110 1115 1120Gly Ser Val Glu Asn
Asn Val Arg Phe Gln Ser Leu Asp Thr Ile Val 1125
1130 1135 Pro Thr Gln Glu Glu Lys Asn Met Val Ile
Asp Phe Glu Leu Asp Ser 1140 1145
1150 Asp Thr Ala His Thr Pro Gln Met Gln Glu Gln Thr Phe Ser Asp
Trp 1155 1160 1165 Trp
Ser Asn Gln Ile Ala Asn Asn Arg Val Val Pro His Tyr Arg Thr 1170
1175 1180 Glu Gly Tyr Phe Met Gln
Phe Thr Arg Asn Thr Ala Ser Ala Val Ser1185 1190
1195 1200His Gln Ile Ala His Asn Glu His Lys Asp Ile
Ile Leu Met Gly Ala 1205 1210
1215 Val Gly Ser Gly Lys Ser Thr Gly Leu Pro Thr Asn Leu Cys Lys Phe
1220 1225 1230 Gly Gly Val
Leu Leu Leu Glu Pro Thr Arg Pro Leu Ala Glu Asn Val 1235
1240 1245 Thr Lys Gln Met Arg Gly Ser Pro
Phe Phe Ala Ser Pro Thr Leu Arg 1250 1255
1260 Met Arg Asn Leu Ser Thr Phe Gly Ser Ser Pro Ile Thr
Val Met Thr1265 1270 1275
1280Thr Gly Phe Ala Leu His Phe Phe Ala Asn Asn Val Lys Glu Phe Asp
1285 1290 1295 Arg Tyr Gln Phe
Ile Ile Phe Asp Glu Phe His Val Leu Asp Ser Asn 1300
1305 1310 Ala Ile Ala Phe Arg Asn Leu Cys His
Glu Tyr Ser Tyr Asn Gly Lys 1315 1320
1325 Ile Ile Lys Val Ser Ala Thr Pro Pro Gly Arg Glu Cys Asp
Leu Thr 1330 1335 1340
Thr Gln Tyr Pro Val Glu Leu Leu Ile Glu Glu Gln Leu Ser Leu Arg1345
1350 1355 1360Asp Phe Val Asp Ala
Gln Gly Thr Asp Ala His Ala Asp Val Val Lys 1365
1370 1375 Lys Gly Asp Asn Ile Leu Val Tyr Val Ala
Ser Tyr Asn Glu Val Asp 1380 1385
1390 Gln Leu Ser Lys Met Leu Asn Glu Arg Gly Phe Leu Val Thr Lys
Val 1395 1400 1405 Asp
Gly Arg Thr Met Lys Leu Gly Gly Val Glu Ile Ile Thr Lys Gly 1410
1415 1420 Ser Ser Ile Lys Lys His
Phe Ile Val Ala Thr Asn Ile Ile Glu Asn1425 1430
1435 1440Gly Val Thr Leu Asp Val Asp Val Val Val Asp
Phe Gly Leu Lys Val 1445 1450
1455 Val Pro Asn Leu Asp Ser Asp Asn Arg Leu Val Ser Tyr Cys Lys Ile
1460 1465 1470 Pro Ile Ser
Leu Gly Glu Arg Ile Gln Arg Phe Gly Arg Val Gly Arg 1475
1480 1485 Asn Lys Pro Gly Val Ala Leu Arg
Ile Gly Glu Thr Ile Lys Gly Leu 1490 1495
1500 Val Glu Ile Pro Ser Met Ile Ala Thr Glu Ala Ala Phe
Leu Cys Phe1505 1510 1515
1520Val Tyr Gly Leu Pro Val Thr Thr Gln Asn Val Ser Thr Ser Ile Leu
1525 1530 1535 Ser Gln Val Ser
Val Arg Gln Ala Arg Val Met Cys Gln Phe Glu Leu 1540
1545 1550 Pro Ile Phe Tyr Thr Ala His Leu Val
Arg Tyr Asp Gly Ala Met His 1555 1560
1565 Pro Ala Ile His Asn Ala Leu Lys Arg Phe Lys Leu Arg Asp
Ser Glu 1570 1575 1580
Ile Asn Leu Asn Thr Leu Ala Ile Pro Thr Ser Ser Ser Lys Thr Trp1585
1590 1595 1600Tyr Thr Gly Lys Cys
Tyr Lys Gln Leu Val Gly Arg Leu Asp Ile Pro 1605
1610 1615 Asp Glu Ile Lys Ile Pro Phe Tyr Thr Lys
Glu Val Pro Glu Lys Val 1620 1625
1630 Pro Glu Gln Ile Trp Asp Val Met Val Lys Phe Ser Ser Asp Ala
Gly 1635 1640 1645 Phe
Gly Arg Met Thr Ser Ala Ala Ala Cys Lys Val Ala Tyr Thr Leu 1650
1655 1660 Gln Thr Asp Ile His Ser
Ile Gln Arg Thr Val Gln Ile Ile Asp Arg1665 1670
1675 1680Leu Leu Glu Asn Glu Met Lys Lys Arg Asn His
Phe Asn Leu Val Val 1685 1690
1695 Asn Gln Ser Cys Ser Ser His Phe Met Ser Leu Ser Ser Ile Met Ala
1700 1705 1710 Ser Leu Arg
Ala His Tyr Ala Lys Asn His Thr Gly Gln Asn Ile Glu 1715
1720 1725 Ile Leu Gln Lys Ala Lys Ala Gln
Leu Leu Glu Phe Ser Asn Leu Ala 1730 1735
1740 Ile Asp Pro Ser Thr Thr Glu Ala Leu Arg Asp Phe Gly
Tyr Leu Glu1745 1750 1755
1760Ala Val Arg Phe Gln Ser Glu Ser Glu Met Ala Arg Gly Leu Lys Leu
1765 1770 1775 Ser Gly His Trp
Lys Trp Ser Leu Ile Ser Arg Asp Leu Ile Val Val 1780
1785 1790 Ser Gly Val Gly Ile Gly Leu Gly Cys
Met Leu Trp Gln Phe Phe Lys 1795 1800
1805 Glu Lys Met His Glu Pro Val Lys Phe Gln Gly Lys Ser Arg
Arg Arg 1810 1815 1820
Leu Gln Phe Arg Lys Ala Arg Asp Asp Lys Met Gly Tyr Ile Met His1825
1830 1835 1840Gly Glu Gly Asp Thr
Ile Glu His Phe Phe Gly Ala Ala Tyr Thr Lys 1845
1850 1855 Lys Gly Lys Ser Lys Gly Lys Thr His Gly
Ala Gly Thr Lys Ala His 1860 1865
1870 Lys Phe Val Asn Met Tyr Gly Val Ser Pro Asp Glu Tyr Ser Tyr
Val 1875 1880 1885 Arg
Tyr Leu Asp Pro Val Thr Gly Ala Thr Leu Asp Glu Ser Pro Met 1890
1895 1900 Thr Asp Leu Asn Ile Val
Gln Glu His Phe Gly Glu Ile Arg Arg Glu1905 1910
1915 1920Ala Ile Leu Ala Asp Ala Met Ser Pro Gln Gln
Arg Asn Lys Gly Ile 1925 1930
1935 Gln Ala Tyr Phe Val Arg Asn Ser Thr Met Pro Ile Leu Lys Val Asp
1940 1945 1950 Leu Thr Pro
His Ile Pro Leu Lys Val Cys Glu Ser Asn Asn Ile Ala 1955
1960 1965 Gly Phe Pro Glu Arg Glu Gly Glu
Leu Arg Arg Thr Gly Pro Thr Glu 1970 1975
1980 Thr Leu Pro Phe Asp Ala Leu Pro Pro Glu Lys Gln Glu
Val Ala Phe1985 1990 1995
2000Glu Ser Lys Ala Leu Leu Lys Gly Val Arg Asp Phe Asn Pro Ile Ser
2005 2010 2015 Ala Cys Val Trp
Leu Leu Glu Asn Ser Ser Asp Gly His Ser Glu Arg 2020
2025 2030 Leu Phe Gly Ile Gly Phe Gly Pro Tyr
Ile Ile Ala Asn Gln His Leu 2035 2040
2045 Phe Arg Arg Asn Asn Gly Glu Leu Thr Ile Lys Thr Met His
Gly Glu 2050 2055 2060
Phe Lys Val Lys Asn Ser Thr Gln Leu Gln Met Lys Pro Val Glu Gly2065
2070 2075 2080Arg Asp Ile Ile Val
Ile Lys Met Ala Lys Asp Phe Pro Pro Phe Pro 2085
2090 2095 Gln Lys Leu Lys Phe Arg Gln Pro Thr Ile
Lys Asp Arg Val Cys Met 2100 2105
2110 Val Ser Thr Asn Phe Gln Gln Lys Ser Val Ser Ser Leu Val Ser
Glu 2115 2120 2125 Ser
Ser His Ile Val His Lys Glu Asp Thr Ser Phe Trp Gln His Trp 2130
2135 2140 Ile Thr Thr Lys Asp Gly
Gln Cys Gly Ser Pro Leu Val Ser Ile Ile2145 2150
2155 2160Asp Gly Asn Ile Leu Gly Ile His Ser Leu Thr
His Thr Thr Asn Gly 2165 2170
2175 Ser Asn Tyr Phe Val Glu Phe Pro Glu Lys Phe Val Ala Thr Tyr Leu
2180 2185 2190 Asp Ala Ala
Asp Gly Trp Cys Lys Asn Trp Lys Phe Asn Ala Asp Lys 2195
2200 2205 Ile Ser Trp Gly Ser Phe Thr Leu
Val Glu Asp Ala Pro Glu Asp Asp 2210 2215
2220 Phe Met Ala Lys Lys Thr Val Ala Ala Ile Met Asp Asp
Leu Val Arg2225 2230 2235
2240Thr Gln Gly Glu Lys Arg Lys Trp Met Leu Glu Ala Ala His Thr Asn
2245 2250 2255 Ile Gln Pro Val
Ala His Leu Gln Ser Gln Leu Val Thr Lys His Ile 2260
2265 2270 Val Lys Gly Arg Cys Lys Met Phe Ala
Leu Tyr Leu Gln Glu Asn Ala 2275 2280
2285 Asp Ala Arg Asp Phe Phe Lys Ser Phe Met Gly Ala Tyr Gly
Pro Ser 2290 2295 2300
His Leu Asn Lys Glu Ala Tyr Ile Lys Asp Ile Met Lys Tyr Ser Lys2305
2310 2315 2320Gln Ile Val Val Gly
Ser Val Asp Cys Asp Thr Phe Glu Ser Ser Leu 2325
2330 2335 Lys Val Leu Ser Arg Lys Met Lys Glu Trp
Gly Phe Glu Asn Leu Glu 2340 2345
2350 Tyr Val Thr Asp Glu Gln Thr Ile Lys Asn Ala Leu Asn Met Asp
Ala 2355 2360 2365 Ala
Val Gly Ala Leu Tyr Ser Gly Lys Lys Lys Gln Tyr Phe Glu Asp 2370
2375 2380 Leu Ser Asp Asp Ala Val
Ala Asn Leu Val Gln Lys Ser Cys Leu Arg2385 2390
2395 2400Leu Phe Lys Asn Lys Leu Gly Val Trp Asn Gly
Ser Leu Lys Ala Glu 2405 2410
2415 Leu Arg Pro Phe Glu Lys Leu Ile Glu Asn Lys Thr Arg Thr Phe Thr
2420 2425 2430 Ala Ala Pro
Ile Glu Thr Leu Leu Gly Gly Lys Val Cys Val Asp Asp 2435
2440 2445 Phe Asn Asn His Phe Tyr Ser Lys
His Ile Gln Cys Pro Trp Ser Val 2450 2455
2460 Gly Met Thr Lys Phe Tyr Gly Gly Trp Asn Glu Leu Leu
Gly Lys Leu2465 2470 2475
2480Pro Asp Gly Trp Val Tyr Cys Asp Ala Asp Gly Ser Gln Phe Asp Ser
2485 2490 2495 Ser Leu Ser Pro
Tyr Leu Ile Asn Ala Val Leu Arg Leu Arg Leu Ser 2500
2505 2510 Ser Met Glu Glu Trp Asp Val Gly Gln
Lys Met Leu Gln Asn Leu Tyr 2515 2520
2525 Thr Glu Ile Val Tyr Thr Pro Ile Ser Thr Pro Asp Gly Thr
Ile Val 2530 2535 2540
Lys Lys Phe Lys Gly Asn Asn Ser Gly Gln Pro Ser Thr Val Val Asp2545
2550 2555 2560Asn Thr Leu Met Val
Val Leu Ala Met Tyr Tyr Ala Leu Ser Lys Leu 2565
2570 2575 Gly Val Asp Ile Asn Ser Gln Glu Asp Val
Cys Lys Phe Phe Ala Asn 2580 2585
2590 Gly Asp Asp Leu Ile Ile Ala Ile Ser Pro Glu Leu Glu His Val
Leu 2595 2600 2605 Asp
Gly Phe Gln Gln His Phe Ser Asp Leu Gly Leu Asn Tyr Asp Phe 2610
2615 2620 Ser Ser Arg Thr Arg Asp
Lys Lys Glu Leu Trp Phe Met Ser His Arg2625 2630
2635 2640Ala Leu Ser Lys Asp Gly Ile Leu Ile Pro Lys
Leu Glu Pro Glu Arg 2645 2650
2655 Ile Val Ser Ile Leu Glu Trp Asp Arg Ser Ala Glu Pro His His Arg
2660 2665 2670 Leu Glu Ala
Ile Cys Ala Ser Met Ile Glu Ala Trp Gly Tyr Thr Asp 2675
2680 2685 Leu Leu Gln Asn Ile Arg Arg Phe
Tyr Lys Trp Thr Ile Glu Gln Glu 2690 2695
2700 Pro Tyr Arg Ser Leu Ala Glu Gln Gly Leu Ala Pro Tyr
Leu Ser Glu2705 2710 2715
2720Val Ala Leu Arg Arg Leu Tyr Thr Ser Gln Ile Ala Thr Asp Asn Glu
2725 2730 2735 Leu Thr Asp Tyr
Tyr Lys Glu Ile Leu Ala Asn Asn Glu Phe Leu Arg 2740
2745 2750 Glu Thr Val Arg Phe Gln Ser Asp Thr
Val Asp Ala Gly Lys Asp Lys 2755 2760
2765 Ala Arg Asp Gln Lys Leu Ala Asp Lys Pro Thr Leu Ala Ile
Asp Arg 2770 2775 2780
Thr Lys Asp Lys Asp Val Asn Thr Gly Thr Ser Gly Thr Phe Ser Ile2785
2790 2795 2800Pro Arg Leu Lys Lys
Ala Ala Met Asn Met Lys Leu Pro Lys Val Gly 2805
2810 2815 Gly Ser Ser Val Val Asn Leu Asp His Leu
Leu Thr Tyr Lys Pro Ala 2820 2825
2830 Gln Glu Phe Val Val Asn Thr Arg Ala Thr His Ser Gln Phe Lys
Ala 2835 2840 2845 Trp
His Thr Asn Val Met Ala Glu Leu Glu Leu Asn Glu Glu Gln Met 2850
2855 2860 Lys Ile Val Leu Asn Gly
Phe Met Ile Trp Cys Ile Glu Asn Gly Thr2865 2870
2875 2880Ser Pro Asn Ile Ser Gly Val Trp Thr Met Met
Asp Gly Asp Glu Gln 2885 2890
2895 Val Glu Tyr Pro Ile Glu Pro Met Val Lys His Ala Asn Pro Ser Leu
2900 2905 2910 Arg Gln Ile
Met Lys His Phe Ser Asn Leu Ala Glu Ala Tyr Ile Arg 2915
2920 2925 Met Arg Asn Ser Glu Gln Val Tyr
Ile Pro Arg Tyr Gly Leu Gln Arg 2930 2935
2940 Gly Leu Val Asp Arg Asn Leu Ala Pro Phe Ala Phe Asp
Phe Phe Glu2945 2950 2955
2960Val Asn Gly Ala Thr Pro Val Arg Ala Arg Glu Ala His Ala Gln Met
2965 2970 2975 Lys Ala Ala Ala
Leu Arg Asn Ser Gln Gln Arg Met Phe Cys Leu Asp 2980
2985 2990 Gly Ser Val Ser Gly Gln Glu Glu Asn
Thr Glu Arg His Thr Val Asp 2995 3000
3005 Asp Val Asn Ala Gln Met His His Leu Leu Gly Val Lys Gly
Val 3010 3015 3020
25381PRTBacillus subtilis 25Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu
Phe Ala Leu Thr Leu1 5 10
15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly Lys
20 25 30 Ser Ser Thr
Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Ser 35
40 45 Ala Met Ser Ser Ala Lys Lys Lys
Asp Val Ile Ser Glu Lys Gly Gly 50 55
60 Lys Val Gln Lys Gln Phe Lys Tyr Val Asn Ala Ala Thr
Ala Thr Leu65 70 75 80
Asp Glu Lys Ala Val Lys Glu Leu Lys Gln Asp Pro Ser Val Ala Tyr
85 90 95 Val Glu Glu Asp His
Ile Ala His Glu Tyr Ala Gln Ser Val Pro Tyr 100
105 110 Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu
His Ser Gln Gly Tyr Thr 115 120
125 Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp
Ser Ser 130 135 140
His Pro Asp Leu Asn Val Lys Gly Gly Ala Ser Phe Val Pro Ser Glu145
150 155 160 Thr Asn Pro Tyr Gln
Asp Gly Ser Ser His Gly Thr His Val Ala Gly 165
170 175 Thr Ile Ala Ala Leu Asn Asn Thr Ile Gly
Val Leu Gly Val Ala Pro 180 185
190 Asn Ala Ser Leu Tyr Ala Val Lys Val Leu Asp Ser Thr Gly Ser
Gly 195 200 205 Gln
Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ser Asn Asn 210
215 220 Met Asp Val Ile Asn Met
Ser Leu Gly Gly Pro Ser Gly Ser Thr Ala225 230
235 240 Leu Lys Thr Val Val Asp Lys Ala Val Ser Ser
Gly Ile Val Val Ala 245 250
255 Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly Ser Thr Ser Thr Val Gly
260 265 270 Tyr Pro Ala
Lys Tyr Pro Ser Thr Ile Ala Val Gly Ala Val Asn Ser 275
280 285 Ser Asn Gln Arg Ala Ser Phe Ser
Ser Ala Gly Ser Glu Leu Asp Val 290 295
300 Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly
Gly Thr Tyr305 310 315
320 Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
325 330 335 Ala Ala Leu Ile
Leu Ser Lys His Pro Thr Trp Thr Asn Ala Gln Val 340
345 350 Arg Asp Arg Leu Glu Ser Thr Ala Thr
Tyr Leu Gly Ser Ser Phe Tyr 355 360
365 Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln
370 375 380 26277PRTRattus norvegicus
26Met Asp Asn Asn Glu Thr Ser Val Asp Ser Lys Ser Ile Asn Asn Phe1
5 10 15 Glu Thr Lys Thr
Ile His Gly Ser Lys Ser Met Asp Ser Gly Ile Tyr 20
25 30 Leu Asp Ser Ser Tyr Lys Met Asp Tyr
Pro Glu Met Gly Leu Cys Ile 35 40
45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Ser
Ala Arg 50 55 60
Asn Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Met Ala65
70 75 80 Leu Lys Tyr Glu Val
Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85
90 95 Met Glu Leu Met Asp Ser Val Ser Lys Glu
Asp His Ser Lys Arg Ser 100 105
110 Ser Phe Val Cys Val Ile Leu Ser His Gly Asp Glu Gly Val Ile
Phe 115 120 125 Gly
Thr Asn Gly Pro Val Asp Leu Lys Lys Leu Thr Ser Phe Phe Arg 130
135 140 Gly Asp Tyr Cys Arg Ser
Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile145 150
155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly
Ile Glu Thr Asp Ser 165 170
175 Gly Thr Asp Asp Asp Met Ala Cys Gln Lys Ile Pro Val Glu Ala Asp
180 185 190 Phe Leu Tyr
Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195
200 205 Ser Arg Asp Gly Ser Trp Phe Ile
Gln Ser Leu Cys Ala Met Leu Lys 210 215
220 Leu Tyr Ala His Lys Leu Glu Phe Met His Ile Leu Thr
Arg Val Asn225 230 235
240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Leu Asp Ala Thr Phe
245 250 255 His Ala Lys Lys
Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260
265 270 Leu Tyr Phe Tyr His 275
277PRTArtificial SequenceTobacco Etch Virus protease cleavage site
consensus sequence 27Glu Xaa Xaa Tyr Xaa Gln Gly1 5
287PRTArtificial SequenceTobacco Etch Virus protease cleavage site
consensus sequence 28Glu Xaa Xaa Tyr Xaa Gln Ser1 5
297PRTArtificial SequenceTobacco Etch Virus protease cleavage site 29Glu
Asn Leu Tyr Phe Gln Gly1 5 307PRTArtificial
SequenceTobacco Etch Virus protease cleavage site 30Glu Asn Leu Tyr Phe
Gln Ser1 5 317PRTArtificial SequenceTobacco Etch
Virus protease cleavage site 31Glu Asn Ile Tyr Thr Gln Gly1
5 327PRTArtificial SequenceTobacco Etch Virus protease cleavage
site 32Glu Asn Ile Tyr Thr Gln Ser1 5
337PRTArtificial SequenceTobacco Etch Virus protease cleavage site 33Glu
Asn Ile Tyr Leu Gln Gly1 5 347PRTArtificial
SequenceTobacco Etch Virus protease cleavage site 34Glu Asn Ile Tyr Leu
Gln Ser1 5 357PRTArtificial SequenceTobacco Etch
Virus protease cleavage site 35Glu Asn Val Tyr Phe Gln Gly1
5 367PRTArtificial SequenceTobacco Etch Virus protease cleavage
site 36Glu Asn Val Tyr Ser Gln Ser1 5
377PRTArtificial SequenceTobacco Etch Virus protease cleavage site 37Glu
Asn Val Tyr Ser Gln Gly1 5 387PRTArtificial
SequenceTobacco Etch Virus protease cleavage site 38Glu Asn Val Tyr Ser
Gln Ser1 5 397PRTArtificial SequenceTobacco Vein
Mottling Virus protease cleavage site consensus sequence 39Xaa Xaa
Val Arg Phe Gln Gly1 5 407PRTArtificial
SequenceTobacco Vein Mottling Virus protease cleavage site consensus
sequence 40Xaa Xaa Val Arg Phe Gln Ser1 5
417PRTArtificial SequenceTobacco Vein Mottling Virus protease cleavage
site 41Glu Thr Val Arg Phe Gln Gly1 5
427PRTArtificial SequenceTobacco Vein Mottling Virus protease cleavage
site 42Glu Thr Val Arg Phe Gln Ser1 5
437PRTArtificial SequenceTobacco Vein Mottling Virus protease cleavage
site 43Asn Asn Val Arg Phe Gln Gly1 5
447PRTArtificial SequenceTobacco Vein Mottling Virus protease cleavage
site 44Asn Asn Val Arg Phe Gln Ser1 5
457PRTArtificial SequenceHuman Rhinovirus 3C protease cleavage site
consensus sequence 45Xaa Xaa Leu Phe Gln Gly Pro1 5
467PRTArtificial SequenceHuman Rhinovirus 3C protease cleavage site
46Glu Ala Leu Phe Gln Gly Pro1 5 477PRTArtificial
SequenceHuman Rhinovirus 3C protease cleavage site 47Glu Val Leu Phe Gln
Gly Pro1 5 487PRTArtificial SequenceHuman
Rhinovirus 3C protease cleavage site 48Glu Leu Leu Phe Gln Gly Pro1
5 497PRTArtificial SequenceHuman Rhinovirus 3C protease
cleavage site 49Asp Ala Leu Phe Gln Gly Pro1 5
507PRTArtificial SequenceHuman Rhinovirus 3C protease cleavage site 50Asp
Val Leu Phe Gln Gly Pro1 5 517PRTArtificial
SequenceHuman Rhinovirus 3C protease cleavage site 51Asp Leu Leu Phe Gln
Gly Pro1 5 526PRTArtificial SequenceSubtilisin
protease cleavage site consensus sequence 52Xaa Xaa Xaa Xaa His Tyr1
5 536PRTArtificial SequenceSubtilisin protease
cleavage site consensus sequence 53Xaa Xaa Xaa Xaa His Tyr1
5 542PRTArtificial SequenceSubtilisin protease cleavage site
54His Tyr1 552PRTArtificial SequenceSubtilisin protease cleavage
site 55Tyr His1 566PRTArtificial SequenceSubtilisin protease
cleavage site 56Pro Gly Ala Ala His Tyr1 5
575PRTArtificial SequenceCaspase 3 protease cleavage site consensus
sequence 57Asp Xaa Xaa Asp Xaa1 5 585PRTArtificial
SequenceCaspase 3 protease cleavage site 58Asp Glu Val Asp Gly1
5 595PRTArtificial SequenceCaspase 3 protease cleavage site 59Asp
Glu Val Asp Ser1 5 605PRTArtificial SequenceCaspase 3
protease cleavage site 60Asp Glu Pro Asp Gly1 5
615PRTArtificial SequenceCaspase 3 protease cleavage site 61Asp Glu Pro
Asp Ser1 5 625PRTArtificial SequenceCaspase 3 protease
cleavage site 62Asp Glu Leu Asp Gly1 5 635PRTArtificial
SequenceCaspase 3 protease cleavage site 63Asp Glu Leu Asp Ser1
5 645PRTArtificial SequenceEnterokinase protease cleavage site
consensus sequence 64Asp Asp Asp Asp Lys1 5
65753DNAArtificial SequenceCodon-optimized TEV open reading frame
65atgggcgaat ctctgttcaa gggtccgcgt gattataacc cgatatcttc tactatttgt
60catctgacta acgaaagcga cggccacacg acttctctgt acggtatcgg tttcggtccg
120ttcatcatta ccaacaagca tctgttccgc cgtaacaacg gtaccctgct ggttcaatct
180ctgcacggcg tcttcaaggt aaaaaatacc actacgctgc agcagcacct gattgacggc
240cgtgacatga tcatcatccg catgccgaaa gattttccgc cgttcccgca aaaactgaag
300tttcgtgaac cgcaacgcga agaacgtatt tgcctggtta ccaccaactt tcagaccaaa
360agcatgtctt ctatggtttc cgatacctct tgcaccttcc caagctctga cggtattttc
420tggaaacatt ggatccagac caaagatggt cagtgcggct ctccgctggt gtctacgcgt
480gacggtttca tcgttggtat ccattctgct tctaacttca ctaacactaa caactacttt
540acttccgttc cgaaaaactt catggagctg ctgactaacc aagaggccca gcagtgggtg
600tccggttggc gcctgaacgc agattctgta ctgtggggtg gtcataaggt attcatgaac
660aaaccggagg agccgttcca gccggtcaaa gaggcgaccc agctgatgaa cgaactggtt
720tactctcagg gtcaccacca tcaccaccat taa
75366250PRTArtificial SequenceTEV protease (S219N) with amino-terminus
polyhistidine affinity tag 66Met Gly Glu Ser Leu Phe Lys Gly Pro Arg
Asp Tyr Asn Pro Ile Ser1 5 10
15 Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
Ser 20 25 30 Leu
Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu 35
40 45 Phe Arg Arg Asn Asn Gly
Thr Leu Leu Val Gln Ser Leu His Gly Val 50 55
60 Phe Lys Val Lys Asn Thr Thr Thr Leu Gln Gln
His Leu Ile Asp Gly65 70 75
80 Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro
85 90 95 Gln Lys Leu
Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu 100
105 110 Val Thr Thr Asn Phe Gln Thr Lys
Ser Met Ser Ser Met Val Ser Asp 115 120
125 Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp
Lys His Trp 130 135 140
Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg145
150 155 160 Asp Gly Phe Ile Val
Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr 165
170 175 Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn
Phe Met Glu Leu Leu Thr 180 185
190 Asn Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala
Asp 195 200 205 Ser
Val Leu Trp Gly Gly His Lys Val Phe Met Asn Lys Pro Glu Glu 210
215 220 Pro Phe Gln Pro Val Lys
Glu Ala Thr Gln Leu Met Asn Glu Leu Val225 230
235 240 Tyr Ser Gln Gly His His His His His His
245 250 67753DNAArtificial
SequenceCodon-optimized TEV open reading frame 67atgggtcacc accatcacca
ccatggcgaa tctctgttca agggtccgcg tgattataac 60ccgatatctt ctactatttg
tcatctgact aacgaaagcg acggccacac gacttctctg 120tacggtatcg gtttcggtcc
gttcatcatt accaacaagc atctgttccg ccgtaacaac 180ggtaccctgg tggttcaatc
tctgcacggc gtcttcaagg taaaaaatac cactacgctg 240cagcagcacc tgattgacgg
ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300ccgttcccgc aaaaactgaa
gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360accaccaact ttcagaccaa
aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420ccaagcggtg acggtatttt
ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480tctccgctgg tgtctacgcg
tgacggtttc atcgttggta tccattctgc ttctaacttc 540actaacacta acaactactt
tacttccgtt ccgaaaaact tcatggagct gctgactaac 600caagaggccc agcagtgggt
gtccggttgg cgcctgaacg cagattctgt actgtggggt 660ggtcataagg tattcatgaa
caaaccggag gagccgttcc agccggtcaa agaggcgacc 720cagctgatga acgaactggt
ttactctcag taa 75368250PRTArtificial
SequenceTEV protease (L56V, S135G, S219N) with amino-terminus
polyhistidine affinity tag 68Met Gly His His His His His His Gly Glu Ser
Leu Phe Lys Gly Pro1 5 10
15 Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu
20 25 30 Ser Asp Gly
His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35
40 45 Ile Ile Thr Asn Lys His Leu Phe
Arg Arg Asn Asn Gly Thr Leu Val 50 55
60 Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr
Thr Thr Leu65 70 75 80
Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro
85 90 95 Lys Asp Phe Pro Pro
Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln 100
105 110 Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
Asn Phe Gln Thr Lys Ser 115 120
125 Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser
Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly145
150 155 160 Ser Pro Leu Val Ser
Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165
170 175 Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
Phe Thr Ser Val Pro Lys 180 185
190 Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val
Ser 195 200 205 Gly
Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210
215 220 Phe Met Asn Lys Pro Glu
Glu Pro Phe Gln Pro Val Lys Glu Ala Thr225 230
235 240 Gln Leu Met Asn Glu Leu Val Tyr Ser Gln
245 250 69753DNAArtificial
SequenceCodon-optimized TEV open reading frame 69atgggtcacc accatcacca
ccatggcgaa tctctgttca agggtccgcg tgattataac 60ccgatatctt cttctatttg
tcatctgact aacgaaagcg acggccacac gacttctctg 120tacggtatcg gtttcggtcc
gttcatcatt accaacaagc atctgttccg ccgtaacaac 180ggtaccctgc tggttcaatc
tctgcacggc gtcttcaagg taaaagacac cactacgctg 240cagcagcacc tggtcgacgg
ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300ccgttcccgc aaaaactgaa
gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360accaccaact ttcagaccaa
aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420ccaagctctg acggtatttt
ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480tctccgctgg tgtctacgcg
tgacggtttc atcgttggta tccattctgc ttctaacttc 540actaacacta acaactactt
tacttccgtt ccgaaaaact tcatggagct gctgactaac 600caagaggccc agcagtgggt
gtccggttgg cgcctgaacg cagattctgt actgtggggt 660ggtcataagg tattcatgaa
caaaccggag gagccgttcc agccggtcaa agaggcgacc 720cagctgatga acgaactggt
ttactctcag taa 75370250PRTArtificial
SequenceTEV protease (T17S, N68D, I77V, S219N) with amino-terminus
polyhistidine affinity tag 70Met Gly His His His His His His Gly Glu Ser
Leu Phe Lys Gly Pro1 5 10
15 Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His Leu Thr Asn Glu
20 25 30 Ser Asp Gly
His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35
40 45 Ile Ile Thr Asn Lys His Leu Phe
Arg Arg Asn Asn Gly Thr Leu Leu 50 55
60 Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asp Thr
Thr Thr Leu65 70 75 80
Gln Gln His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro
85 90 95 Lys Asp Phe Pro Pro
Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln 100
105 110 Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
Asn Phe Gln Thr Lys Ser 115 120
125 Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser
Ser Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly145
150 155 160 Ser Pro Leu Val Ser
Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165
170 175 Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
Phe Thr Ser Val Pro Lys 180 185
190 Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val
Ser 195 200 205 Gly
Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210
215 220 Phe Met Asn Lys Pro Glu
Glu Pro Phe Gln Pro Val Lys Glu Ala Thr225 230
235 240 Gln Leu Met Asn Glu Leu Val Tyr Ser Gln
245 250 71753DNAArtificial
SequenceCodon-optimized TEV open reading frame 71atgggtcacc accatcacca
ccatggcgaa tctctgttca agggtccgcg tgattataac 60ccgatatctt ctactatttg
tcatctgact aacgaaagcg acggccacac gacttctctg 120tacggtatcg gtttcggtcc
gttcatcatt accgtgaagc atctgttccg ccgtaacaac 180ggtaccctgg tggttcaatc
tctgcacggc gtcttcaagg taaaaaatac cactacgctg 240cagcagcacc tgattgacgg
ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300ccgttcccgc aaaaactgaa
gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360accaccaact ttcagaccaa
aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420ccaagcggtg acggtatttt
ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480tctccgctgg tgtctacgcg
tgacggtttc atcgttggta tccattctgc ttctaacttc 540actaacacta acaactactt
tacttccgtt ccgaaaaact tcatggagct gctgactaac 600caagaggccc agcagtgggt
gtccggttgg cgcctgaacg cagattctgt actgtggggt 660ggtcataagg tattcatgaa
caaaccggag gagccgttcc agccggtcaa agaggcgacc 720cagctgatga acgaactggt
ttactctcag taa 75372250PRTArtificial
SequenceTEV protease (N44V, L56V, S135G, S219N) with amino-terminus
polyhistidine affinity tag 72Met Gly His His His His His His Gly Glu Ser
Leu Phe Lys Gly Pro1 5 10
15 Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu
20 25 30 Ser Asp Gly
His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35
40 45 Ile Ile Thr Val Lys His Leu Phe
Arg Arg Asn Asn Gly Thr Leu Val 50 55
60 Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr
Thr Thr Leu65 70 75 80
Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro
85 90 95 Lys Asp Phe Pro Pro
Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln 100
105 110 Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
Asn Phe Gln Thr Lys Ser 115 120
125 Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser
Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly145
150 155 160 Ser Pro Leu Val Ser
Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165
170 175 Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
Phe Thr Ser Val Pro Lys 180 185
190 Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val
Ser 195 200 205 Gly
Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210
215 220 Phe Met Asn Lys Pro Glu
Glu Pro Phe Gln Pro Val Lys Glu Ala Thr225 230
235 240 Gln Leu Met Asn Glu Leu Val Tyr Ser Gln
245 250 73753DNAArtificial
SequenceCodon-optimized TEV open reading frame 73atgggtcacc accatcacca
ccatggcgaa tctctgttca agggtccgcg tgattataac 60ccgatatctt ctactatttg
tcatctgact aacgaaagcg acggccacac gacttctctg 120tacggtatcg gtttcggtcc
gttcatcatt accaacaagc atctgttccg ccgtaacaac 180ggtaccctgg tggttcaatc
tctgcacggc gtcttcaagg taaaagacac cactacgctg 240cagcagcacc tgattgacgg
ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300ccgttcccgc aaaaactgaa
gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360accaccaact ttcagaccaa
aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420ccaagcggtg acggtatttt
ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480tctccgctgg tgtctacgcg
tgacggtttc atcgttggta tccattctgc ttctaacttc 540actaacacta acaactactt
tacttccgtt ccgaaaaact tcatggagct gctgactaac 600caagaggccc agcagtgggt
gtccggttgg cgcctgaacg cagattctgt actgtggggt 660ggtcataagg tattcatgaa
caaaccggag gagccgttcc agccggtcaa agaggcgacc 720cagctgatga acgaactggt
ttactctcag taa 75374250PRTArtificial
SequenceTEV protease (L56V, N68D, S135G, S219N) with amino-terminus
polyhistidine affinity tag 74Met Gly His His His His His His Gly Glu Ser
Leu Phe Lys Gly Pro1 5 10
15 Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu
20 25 30 Ser Asp Gly
His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe 35
40 45 Ile Ile Thr Asn Lys His Leu Phe
Arg Arg Asn Asn Gly Thr Leu Val 50 55
60 Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asp Thr
Thr Thr Leu65 70 75 80
Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro
85 90 95 Lys Asp Phe Pro Pro
Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln 100
105 110 Arg Glu Glu Arg Ile Cys Leu Val Thr Thr
Asn Phe Gln Thr Lys Ser 115 120
125 Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser
Gly Asp 130 135 140
Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly145
150 155 160 Ser Pro Leu Val Ser
Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser 165
170 175 Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr
Phe Thr Ser Val Pro Lys 180 185
190 Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val
Ser 195 200 205 Gly
Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val 210
215 220 Phe Met Asn Lys Pro Glu
Glu Pro Phe Gln Pro Val Lys Glu Ala Thr225 230
235 240 Gln Leu Met Asn Glu Leu Val Tyr Ser Gln
245 250 75753DNAArtificial
SequenceCodon-optimized TEV open reading frame 75atgggtcacc accatcacca
ccatggcgaa tctctgttca agggtccgcg tgattataac 60ccgatatctt cttctatttg
tcatctgact aacgaaagcg acggccacac gacttctctg 120tacggtatcg gtttcggtcc
gttcatcatt accaacaagc atctgttccg ccgtaacaac 180ggtaccctgg tggttcaatc
tctgcacggc gtcttcaagg taaaagacac cactacgctg 240cagcagcacc tggtcgacgg
ccgtgacatg atcatcatcc gcatgccgaa agattttccg 300ccgttcccgc aaaaactgaa
gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt 360accaccaact ttcagaccaa
aagcatgtct tctatggttt ccgatacctc ttgcaccttc 420ccaagctctg acggtatttt
ctggaaacat tggatccaga ccaaagatgg tcagtgcggc 480tctccgctgg tgtctacgcg
tgacggtttc atcgttggta tccattctgc ttctaacttc 540actaacacta acaactactt
tacttccgtt ccgaaaaact tcatggagct gctgactaac 600caagaggccc agcagtgggt
gtccggttgg cgcctgaacg cagattctgt actgtggggt 660ggtcataagg tattcatgaa
caaaccggag gagccgttcc agccggtcaa agaggcgacc 720cagctgatga acgaactggt
ttactctcag taa 75376250PRTArtificial
SequenceTEV protease (T17S, L56V, N68D, I77V, S219N) with
amino-terminus polyhistidine affinity tag 76Met Gly His His His His His
His Gly Glu Ser Leu Phe Lys Gly Pro1 5 10
15 Arg Asp Tyr Asn Pro Ile Ser Ser Ser Ile Cys His
Leu Thr Asn Glu 20 25 30
Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe
35 40 45 Ile Ile Thr Asn
Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val 50 55
60 Val Gln Ser Leu His Gly Val Phe Lys
Val Lys Asp Thr Thr Thr Leu65 70 75
80 Gln Gln His Leu Val Asp Gly Arg Asp Met Ile Ile Ile Arg
Met Pro 85 90 95
Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln
100 105 110 Arg Glu Glu Arg Ile
Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser 115
120 125 Met Ser Ser Met Val Ser Asp Thr Ser
Cys Thr Phe Pro Ser Ser Asp 130 135
140 Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly
Gln Cys Gly145 150 155
160 Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser
165 170 175 Ala Ser Asn Phe
Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180
185 190 Asn Phe Met Glu Leu Leu Thr Asn Gln
Glu Ala Gln Gln Trp Val Ser 195 200
205 Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His
Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr225
230 235 240 Gln Leu Met Asn Glu
Leu Val Tyr Ser Gln 245 250
77753DNAArtificial SequenceCodon-optimized TEV open reading frame
77atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac
60ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg
120tacggtatcg gtttcggtcc gttcatcatt accaacaagc atctgttccg ccgtaacaac
180ggtaccctgc tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg
240cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg
300ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt
360accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc
420ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc
480tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc
540actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac
600caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt
660ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc
720cagctgatga acgaactggt ttactctcag taa
75378250PRTArtificial SequenceTEV protease (T17S, N68D, I77V, S135G,
S219N) with amino-terminus polyhistidine affinity tag 78Met Gly His
His His His His His Gly Glu Ser Leu Phe Lys Gly Pro1 5
10 15 Arg Asp Tyr Asn Pro Ile Ser Ser
Ser Ile Cys His Leu Thr Asn Glu 20 25
30 Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe
Gly Pro Phe 35 40 45
Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu 50
55 60 Val Gln Ser Leu His
Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu65 70
75 80 Gln Gln His Leu Val Asp Gly Arg Asp Met
Ile Ile Ile Arg Met Pro 85 90
95 Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro
Gln 100 105 110 Arg
Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser 115
120 125 Met Ser Ser Met Val Ser
Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130 135
140 Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys
Asp Gly Gln Cys Gly145 150 155
160 Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser
165 170 175 Ala Ser Asn
Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys 180
185 190 Asn Phe Met Glu Leu Leu Thr Asn
Gln Glu Ala Gln Gln Trp Val Ser 195 200
205 Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly
His Lys Val 210 215 220
Phe Met Asn Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr225
230 235 240 Gln Leu Met Asn Glu
Leu Val Tyr Ser Gln 245 250
79753DNAArtificial SequenceCodon-optimized TEV open reading frame
79atgggcgaat ctctgttcaa gggtccgcgt gattataacc cgatatcttc ttctatttgt
60catctgacta acgaaagcga cggccacacg acttctctgt acggtatcgg tttcggtccg
120ttcatcatta ccgtgaagca tctgttccgc cgtaacaacg gtaccctggt ggttcaatct
180ctgcacggcg tcttcaaggt aaaagacacc actacgctgc agcagcacct ggtcgacggc
240cgtgacatga tcatcatccg catgccgaaa gattttccgc cgttcccgca aaaactgaag
300tttcgtgaac cgcaacgcga agaacgtatt tgcctggtta ccaccaactt tcagaccaaa
360agcatgtctt ctatggtttc cgatacctct tgcaccttcc caagcggtga cggtattttc
420tggaaacatt ggatccagac caaagatggt cagtgcggct ctccgctggt gtctacgcgt
480gacggtttca tcgttggtat ccattctgct tctaacttca ctaacactaa caactacttt
540acttccgttc cgaaaaactt catggagctg ctgactaacc aagaggccca gcagtgggtg
600tccggttggc gcctgaacgc agattctgta ctgtggggtg gtcataaggt attcatgaac
660aaaccggagg agccgttcca gccggtcaaa gaggcgaccc agctgatgaa cgaactggtt
720tactctcagg gtcaccacca tcaccaccat taa
75380250PRTArtificial SequenceTEV protease (T17S, N44V, L56V, N68D, I77V,
S135G, S219N) with carboxyl-terminus polyhistidine affinity tag
80Met Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser1
5 10 15 Ser Ser Ile Cys
His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser 20
25 30 Leu Tyr Gly Ile Gly Phe Gly Pro Phe
Ile Ile Thr Val Lys His Leu 35 40
45 Phe Arg Arg Asn Asn Gly Thr Leu Val Val Gln Ser Leu His
Gly Val 50 55 60
Phe Lys Val Lys Asp Thr Thr Thr Leu Gln Gln His Leu Val Asp Gly65
70 75 80 Arg Asp Met Ile Ile
Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro 85
90 95 Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg
Glu Glu Arg Ile Cys Leu 100 105
110 Val Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser
Asp 115 120 125 Thr
Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp 130
135 140 Ile Gln Thr Lys Asp Gly
Gln Cys Gly Ser Pro Leu Val Ser Thr Arg145 150
155 160 Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser
Asn Phe Thr Asn Thr 165 170
175 Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr
180 185 190 Asn Gln Glu
Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp 195
200 205 Ser Val Leu Trp Gly Gly His Lys
Val Phe Met Asn Lys Pro Glu Glu 210 215
220 Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn
Glu Leu Val225 230 235
240 Tyr Ser Gln Gly His His His His His His 245
250 81753DNAArtificial SequenceCodon-optimized TEV open reading
frame 81atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac
60ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg
120tacggtatcg gtttcggtcc gttcatcatt accgtgaagc atctgttccg ccgtaacaac
180ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg
240cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg
300ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt
360accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc
420ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc
480tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc
540actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac
600caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt
660ggtcataagg tattcatggt gaaaccggag gagccgttcc agccggtcaa agaggcgacc
720cagctgatga acgaactggt ttactctcag taa
75382250PRTArtificial SequenceTEV protease (T17S, N44V, L56V, N68D, I77V,
S135G, S219V) with amino-terminus polyhistidine affinity tag 82Met
Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro1
5 10 15 Arg Asp Tyr Asn Pro Ile
Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25
30 Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile
Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Val Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val
50 55 60 Val Gln Ser
Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu65 70
75 80 Gln Gln His Leu Val Asp Gly Arg
Asp Met Ile Ile Ile Arg Met Pro 85 90
95 Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg
Glu Pro Gln 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser
115 120 125 Met Ser Ser Met
Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130
135 140 Gly Ile Phe Trp Lys His Trp Ile
Gln Thr Lys Asp Gly Gln Cys Gly145 150
155 160 Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val
Gly Ile His Ser 165 170
175 Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys
180 185 190 Asn Phe Met
Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser 195
200 205 Gly Trp Arg Leu Asn Ala Asp Ser
Val Leu Trp Gly Gly His Lys Val 210 215
220 Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val Lys
Glu Ala Thr225 230 235
240 Gln Leu Met Asn Glu Leu Val Tyr Ser Gln 245
250 83753DNAArtificial SequenceCodon-optimized TEV open reading
frame 83atgggtcacc accatcacca ccatggcgaa tctctgttca agggtccgcg tgattataac
60ccgatatctt cttctatttg tcatctgact aacgaaagcg acggccacac gacttctctg
120tacggtatcg gtttcggtcc gttcatcatt accgtgaagc atctgttccg ccgtaacaac
180ggtaccctgg tggttcaatc tctgcacggc gtcttcaagg taaaagacac cactacgctg
240cagcagcacc tggtcgacgg ccgtgacatg atcatcatcc gcatgccgaa agattttccg
300ccgttcccgc aaaaactgaa gtttcgtgaa ccgcaacgcg aagaacgtat ttgcctggtt
360accaccaact ttcagaccaa aagcatgtct tctatggttt ccgatacctc ttgcaccttc
420ccaagcggtg acggtatttt ctggaaacat tggatccaga ccaaagatgg tcagtgcggc
480tctccgctgg tgtctacgcg tgacggtttc atcgttggta tccattctgc ttctaacttc
540actaacacta acaactactt tacttccgtt ccgaaaaact tcatggagct gctgactaac
600caagaggccc agcagtgggt gtccggttgg cgcctgaacg cagattctgt actgtggggt
660ggtcataagg tattcatgaa caaaccggag gagccgttcc agccggtcaa agaggcgacc
720cagctgatga acgaactggt ttactctcag taa
75384250PRTArtificial SequenceTEV protease (T17S, N44V, L56V, N68D, I77V,
S135G, S219N) with amino-terminus polyhistidine affinity tag 84Met
Gly His His His His His His Gly Glu Ser Leu Phe Lys Gly Pro1
5 10 15 Arg Asp Tyr Asn Pro Ile
Ser Ser Ser Ile Cys His Leu Thr Asn Glu 20 25
30 Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile
Gly Phe Gly Pro Phe 35 40 45
Ile Ile Thr Val Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Val
50 55 60 Val Gln Ser
Leu His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu65 70
75 80 Gln Gln His Leu Val Asp Gly Arg
Asp Met Ile Ile Ile Arg Met Pro 85 90
95 Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg
Glu Pro Gln 100 105 110
Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser
115 120 125 Met Ser Ser Met
Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Gly Asp 130
135 140 Gly Ile Phe Trp Lys His Trp Ile
Gln Thr Lys Asp Gly Gln Cys Gly145 150
155 160 Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val
Gly Ile His Ser 165 170
175 Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys
180 185 190 Asn Phe Met
Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser 195
200 205 Gly Trp Arg Leu Asn Ala Asp Ser
Val Leu Trp Gly Gly His Lys Val 210 215
220 Phe Met Asn Lys Pro Glu Glu Pro Phe Gln Pro Val Lys
Glu Ala Thr225 230 235
240 Gln Leu Met Asn Glu Leu Val Tyr Ser Gln 245
250 85750DNAArtificial SequenceNative TEV open reading frame
85atgcatcacc atcaccacca tggagaaagc ttgtttaagg gaccacgtga ttacaacccg
60atatcgagca ccatttgtca tttgacgaat gaatctgatg ggcacacaac atcgttgtat
120ggtattggat ttggtccctt catcattaca aacaagcact tgtttcgccg taataatgga
180acactgttgg tccaatcact acatggtgta ttcaaggtca agaacaccac gactttgcaa
240caacacctca ttgatgggag ggacatgata attattcgca tgcctaagga tttcccacca
300tttcctcaaa agctgaaatt tagagagcca caaagggaag agcgcatctg tcttgtgaca
360accaacttcc aaactaagag catgtctagc atggtgtcag acactagttg cacattccct
420tcatctgatg gcatattctg gaagcattgg atccaaacca aggatgggca gtgtggcagt
480ccattagtat caactagaga tgggttcatt gttggtatac actcagcatc gaatttcacc
540aacacaaaca attatttcac aagcgtgccg aaaaacttca tggaattgtt gacaaatcag
600gaggcgcagc agtgggttag tggttggcga ttaaatgctg actcagtatt gtgggggggc
660cataaagttt tcatgaacaa acctgaagag ccttttcagc cagttaagga agcgactcaa
720ctcatgaatg aattggtgta ctcgcaataa
75086249PRTArtificial SequenceTEV protease (S219N) with amino-terminus
polyhistidine affinity tag 86Met His His His His His His Gly Glu Ser
Leu Phe Lys Gly Pro Arg1 5 10
15 Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu
Ser 20 25 30 Asp
Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile 35
40 45 Ile Thr Asn Lys His Leu
Phe Arg Arg Asn Asn Gly Thr Leu Leu Val 50 55
60 Gln Ser Leu His Gly Val Phe Lys Val Lys Asn
Thr Thr Thr Leu Gln65 70 75
80 Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys
85 90 95 Asp Phe Pro
Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg 100
105 110 Glu Glu Arg Ile Cys Leu Val Thr
Thr Asn Phe Gln Thr Lys Ser Met 115 120
125 Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser
Ser Asp Gly 130 135 140
Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser145
150 155 160 Pro Leu Val Ser Thr
Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala 165
170 175 Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe
Thr Ser Val Pro Lys Asn 180 185
190 Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser
Gly 195 200 205 Trp
Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe 210
215 220 Met Asn Lys Pro Glu Glu
Pro Phe Gln Pro Val Lys Glu Ala Thr Gln225 230
235 240 Leu Met Asn Glu Leu Val Tyr Ser Gln
245 873984DNAArtificial SequenceOpen reading frame
encoding BoNT/A-TEV 87atgccgttcg taaacaaaca gttcaactat aaagacccag
tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga
aagcatttaa aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg tcttactatg
atagcacata cctgagtacc 240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac
tgttcgagcg catttattcg 300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg
gcattccgtt ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact
gcatcaatgt gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat gaagtcctga
atctgacgcg gaatggctat 540ggatcgacgc agtatattcg tttttctcca gatttcacat
ttggatttga agaaagcctc 600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg
cgacggaccc agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg
gaatcgccat taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat gcaaaattta
ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta caataaattc aaagacattg
catcaacctt aaacaaggcg 900aaaagcattg tgggtaccac ggctagctta caatatatga
aaaacgtttt caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg
ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat aaagccgtct
ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta tgatggcttt aatctgcgca
atacgaatct ggcggcgaac 1200tttaacggcc agaacaccga aatcaacaac atgaacttta
ctaaactgaa aaattttacc 1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta
ttatcaccag caaaaccaaa 1320tccttgggcg gtggtggcga aaacctgtac ttccagggcg
gtggcggtgg tgataagggc 1380tataacaagg ccttaaatga tttatgcatc aaggtgaaca
actgggactt gtttttctct 1440ccatctgaag ataattttac taacgacttg aacaaaggag
aggaaattac ttccgatacc 1500aacatcgaag cagcggaaga gaatattagc ctggatctta
ttcaacaata ttacctgacc 1560tttaattttg ataacgagcc tgagaacatt tccattgaga
atctcagctc tgacatcatc 1620ggccagctgg aactgatgcc gaatatcgaa cgctttccta
atggaaagaa atatgaattg 1680gacaaataca ccatgttcca ctatctccgc gcgcaggagt
ttgagcacgg caagtctcgt 1740attgctctga ccaattcggt aaacgaagcc cttttaaatc
cttcgcgtgt gtacaccttt 1800ttctcaagcg attatgttaa aaaagtgaac aaggcgaccg
aagcggcgat gtttttggga 1860tgggtggaac aactggtata tgactttacg gatgaaactt
ctgaagtctc gaccaccgac 1920aaaattgccg atattaccat tatcattccc tatattggcc
ctgcactgaa cattggtaac 1980atgctgtata aagatgattt tgtgggcgcc ctgatctttt
caggcgctgt tatcctgctg 2040gaatttatcc cggaaatcgc cattccagta ctcggtacct
ttgcgctggt gtcctatatc 2100gcaaacaaag ttttgactgt ccagacgatc gacaacgcgc
tcagtaaacg taacgaaaaa 2160tgggatgagg tgtataagta tattgttacc aactggctcg
ctaaagtaaa cacccagatt 2220gacctgattc gcaagaagat gaaagaagcg ctggaaaacc
aagcagaagc gaccaaagct 2280attatcaact atcaatataa ccagtacaca gaggaagaaa
agaataacat caacttcaac 2340atcgacgact tatcttcaaa gctgaatgaa tctattaaca
aagcgatgat taatattaac 2400aagttcttga accaatgtag tgtcagctat ctgatgaact
cgatgatccc ttacggtgtg 2460aaacgtctgg aagacttcga tgcaagcctt aaagatgccc
ttctgaagta tatttacgat 2520aatcgcggaa ctcttattgg ccaagtggat cgcttaaaag
ataaagtcaa caacacgctg 2580agtacagaca tcccttttca gctgtctaaa tatgtggaca
atcagcgcct gctgtccacg 2640tttacggaat acatcaaaaa catcatcaac actagtattc
tgaacttgcg ttacgagagt 2700aaccatctga ttgatctgag ccgttacgca tctaaaatca
acatcggctc gaaggtgaac 2760ttcgatccta tcgacaaaaa ccagattcaa ttgttcaact
tagaatcgtc aaagattgaa 2820gttatcttaa aaaatgcgat tgtatataat tcaatgtacg
aaaatttctc tacgagcttt 2880tggattcgta ttccgaaata tttcaacagt atctctttaa
acaacgagta tactatcatc 2940aattgtatgg agaataacag cgggtggaaa gtgagcctta
actatggtga aatcatctgg 3000actctgcagg acactcaaga aattaaacaa cgcgtggtgt
ttaaatactc acagatgatt 3060aacatctcgg attatattaa tcgctggatt tttgtgacaa
ttactaacaa ccggctgaac 3120aacagcaaaa tttacattaa cggtcgcctg atcgatcaga
aaccaatcag taatctcggt 3180aacattcacg catcgaataa tatcatgttc aaactggatg
gttgtcgcga cacgcaccgt 3240tacatttgga tcaaatactt caatttattc gacaaagaac
tcaacgaaaa ggagattaag 3300gatctttatg acaatcagtc taattcgggt attctgaaag
acttttgggg tgattacctt 3360cagtacgata aaccgtatta tatgttaaac ttatatgatc
cgaataaata cgttgacgtc 3420aacaacgttg gcattcgtgg ctatatgtat ctgaaagggc
cgcgtggcag cgtgatgacc 3480actaacattt acttaaactc ctccctctat cgcggtacta
aatttattat caagaaatat 3540gcctctggca acaaggacaa tatcgtacgc aataacgatc
gcgtctacat taacgtggtg 3600gtgaagaata aagaatatcg tctggcgacc aatgctagtc
aggcgggcgt ggagaaaatt 3660ctgtctgcac ttgaaatccc ggatgtgggt aatttatccc
aggtggttgt gatgaaaagt 3720aaaaatgacc aagggatcac caataaatgc aaaatgaatc
tgcaagataa caacggcaac 3780gacattggtt ttatcggctt ccaccaattc aataatatcg
cgaaactggt ggcctcaaat 3840tggtacaacc gtcagattga gcgcagctcc cgcactttag
gctgtagctg ggagttcatt 3900ccggtagatg acggttgggg agaacgccca ttgaaagtcg
acaagcttgc ggccgcactc 3960gagcaccacc accaccacca ctga
3984881327PRTArtificial SequenceBoNT/A-TEV 88Met
Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1
5 10 15 Val Asp Ile Ala Tyr Ile
Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25
30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp
Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu
50 55 60 Ala Lys Gln
Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70
75 80 Asp Asn Glu Lys Asp Asn Tyr Leu
Lys Gly Val Thr Lys Leu Phe Glu 85 90
95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr
Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys
115 120 125 Val Ile Asp Thr
Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130
135 140 Arg Ser Glu Glu Leu Asn Leu Val
Ile Ile Gly Pro Ser Ala Asp Ile145 150
155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val
Leu Asn Leu Thr 165 170
175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe
180 185 190 Thr Phe Gly
Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195
200 205 Gly Ala Gly Lys Phe Ala Thr Asp
Pro Ala Val Thr Leu Ala His Glu 210 215
220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn Pro Asn225 230 235
240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu
245 250 255 Glu Val Ser Phe
Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260
265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu
Phe Arg Leu Tyr Tyr Tyr Asn 275 280
285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser
Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305
310 315 320 Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325
330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr Glu Asp 340 345
350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu
Asn 355 360 365 Phe
Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370
375 380 Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390
395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met
Asn Phe Thr Lys Leu 405 410
415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg
420 425 430 Gly Ile Ile
Thr Ser Lys Thr Lys Ser Leu Gly Gly Gly Gly Glu Asn 435
440 445 Leu Tyr Phe Gln Gly Gly Gly Gly
Gly Asp Lys Gly Tyr Asn Lys Ala 450 455
460 Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu
Phe Phe Ser465 470 475
480 Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile
485 490 495 Thr Ser Asp Thr
Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp 500
505 510 Leu Ile Gln Gln Tyr Tyr Leu Thr Phe
Asn Phe Asp Asn Glu Pro Glu 515 520
525 Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln
Leu Glu 530 535 540
Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu545
550 555 560 Asp Lys Tyr Thr Met
Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His 565
570 575 Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser
Val Asn Glu Ala Leu Leu 580 585
590 Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys
Lys 595 600 605 Val
Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln 610
615 620 Leu Val Tyr Asp Phe Thr
Asp Glu Thr Ser Glu Val Ser Thr Thr Asp625 630
635 640 Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr
Ile Gly Pro Ala Leu 645 650
655 Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile
660 665 670 Phe Ser Gly
Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala Ile 675
680 685 Pro Val Leu Gly Thr Phe Ala Leu
Val Ser Tyr Ile Ala Asn Lys Val 690 695
700 Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg
Asn Glu Lys705 710 715
720 Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val
725 730 735 Asn Thr Gln Ile
Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu Glu 740
745 750 Asn Gln Ala Glu Ala Thr Lys Ala Ile
Ile Asn Tyr Gln Tyr Asn Gln 755 760
765 Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp
Asp Leu 770 775 780
Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn785
790 795 800 Lys Phe Leu Asn Gln
Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile 805
810 815 Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe
Asp Ala Ser Leu Lys Asp 820 825
830 Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly
Gln 835 840 845 Val
Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp Ile 850
855 860 Pro Phe Gln Leu Ser Lys
Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr865 870
875 880 Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr
Ser Ile Leu Asn Leu 885 890
895 Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys
900 905 910 Ile Asn Ile
Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln 915
920 925 Ile Gln Leu Phe Asn Leu Glu Ser
Ser Lys Ile Glu Val Ile Leu Lys 930 935
940 Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser
Thr Ser Phe945 950 955
960 Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu
965 970 975 Tyr Thr Ile Ile
Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser 980
985 990 Leu Asn Tyr Gly Glu Ile Ile Trp Thr
Leu Gln Asp Thr Gln Glu Ile 995 1000
1005 Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile
Ser Asp 1010 1015 1020
Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn1025
1030 1035 1040Asn Ser Lys Ile Tyr
Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile 1045
1050 1055 Ser Asn Leu Gly Asn Ile His Ala Ser Asn
Asn Ile Met Phe Lys Leu 1060 1065
1070 Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe
Asn 1075 1080 1085 Leu
Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp 1090
1095 1100 Asn Gln Ser Asn Ser Gly
Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu1105 1110
1115 1120Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu
Tyr Asp Pro Asn Lys 1125 1130
1135 Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys
1140 1145 1150 Gly Pro Arg
Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser 1155
1160 1165 Leu Tyr Arg Gly Thr Lys Phe Ile
Ile Lys Lys Tyr Ala Ser Gly Asn 1170 1175
1180 Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile
Asn Val Val1185 1190 1195
1200Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly
1205 1210 1215 Val Glu Lys Ile
Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu 1220
1225 1230 Ser Gln Val Val Val Met Lys Ser Lys
Asn Asp Gln Gly Ile Thr Asn 1235 1240
1245 Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile
Gly Phe 1250 1255 1260
Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn1265
1270 1275 1280Trp Tyr Asn Arg Gln
Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser 1285
1290 1295 Trp Glu Phe Ile Pro Val Asp Asp Gly Trp
Gly Glu Arg Pro Leu Lys 1300 1305
1310 Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His
1315 1320 1325
891044DNAArtificial SequenceIntervening sequence containing
transcriptional and translational sites. 89aagcttgtgg cctcaaattg
gtacaaccgt cagattgagc gcagctcccg cactttaggc 60tgtagctggg agttcattcc
ggtagatgac ggttggggag aacgcccatt gcaccatcat 120caccatcact gagcggccgc
ataatgctta agtcgaacag attgatatgt agctataagt 180aatcgtattg tacacggccg
cataatcgaa attaatacga ctcactatag gggaattgtg 240agcggataac aattccccat
cttagtatat tagttaagta taagaaggag atataccatg 300ggcgaatctc tgttcaaggg
tccgcgtgat tataacccga tatcttcttc tatttgtcat 360ctgactaacg aaagcgacgg
ccacacgact tctctgtacg gtatcggttt cggtccgttc 420atcattacca acaagcatct
gttccgccgt aacaacggta ccctgctggt tcaatctctg 480cacggcgtct tcaaggtaaa
agacaccact acgctgcagc agcacctggt cgacggccgt 540gacatgatca tcatccgcat
gccgaaagat tttccgccgt tcccgcaaaa actgaagttt 600cgtgaaccgc aacgcgaaga
acgtatttgc ctggttacca ccaactttca gaccaaaagc 660atgtcttcta tggtttccga
tacctcttgc accttcccaa gcggtgacgg tattttctgg 720aaacattgga ttcagaccaa
agatggtcag tgcggctctc cgctggtgtc tacgcgtgac 780ggtttcatcg ttggtatcca
ttctgcttct aacttcacta acactaacaa ctactttact 840tccgttccga aaaacttcat
ggagctgctg actaaccaag aggcccagca gtgggtgtcc 900ggttggcgcc tgaacgcaga
ttctgtactg tggggtggtc ataaggtatt catgaacaaa 960ccggaggagc cgttccagcc
ggtcaaagag gcgacccagc tgatgaacga actggtttac 1020tctcagtaag agctctgtct
cgag 1044904851DNAArtificial
SequenceBoNT/A-TEV and TEV protease variant 4 open reading frames
with the intervening transcription and translation elements.
90atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt ggacattgcc
60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa aatccataac
120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg agatttaaac
180ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata cctgagtacc
240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg catttattcg
300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt ttggggtggt
360agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt gattcagcct
420gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag cgcggatatt
480atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg gaatggctat
540ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga agaaagcctc
600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc agcggtgacc
660ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat taacccgaac
720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga agtgtccttt
780gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct gcaagaaaac
840gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt aaacaaggcg
900aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt caaagaaaaa
960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa atttgataaa
1020ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt cttcaaggtc
1080ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa catcgtaccg
1140aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct ggcggcgaac
1200tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa aaattttacc
1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta ttatcaccag caaaaccaaa
1320tccttgggcg gtggtggcga aaacctgtac ttccagggcg gtggcggtgg tgataagggc
1380tataacaagg ccttcaatga tttatgcatc aaggtgaaca actgggactt gtttttctct
1440ccatctgaag ataattttac taacgacttg aacaaaggag aggaaattac ttccgatacc
1500aacatcgaag cagcggaaga gaatattagt ctagatctta ttcaacaata ttacctgacc
1560tttaattttg ataacgagcc tgagaacatt tccattgaga atctcagctc tgacatcatc
1620ggccagctgg aactgatgcc gaatatcgaa cgctttccta atggaaagaa atatgaattg
1680gacaaataca ccatgttcca ctatctccgc gcgcaggagt ttgagcacgg caagtctcgt
1740attgctctga ccaattcggt aaacgaagcc cttttaaatc cttcgcgtgt gtacaccttt
1800ttctcaagcg attatgttaa aaaagtgaac aaggcgaccg aagcggcgat gtttttggga
1860tgggtggaac aactggtata tgactttacg gatgaaactt ctgaagtctc gaccaccgac
1920aaaattgccg atattaccat tatcattccc tatattggcc ctgcactgaa cattggtaac
1980atgctgtata aagatgattt tgtgggcgcc ctgatctttt caggcgctgt tatcctgctg
2040gaatttatcc cggaaatcgc cattccagta ctcggtacct ttgcgctggt gtcctatatc
2100gcaaacaaag ttttgactgt ccagacgatc gacaacgcgc tcagtaaacg taacgaaaaa
2160tgggatgagg tgtataagta tattgttacc aactggctcg ctaaagtaaa cacccagatt
2220gacctgattc gcaagaagat gaaagaagcg ctggaaaacc aagcagaagc gaccaaagct
2280attatcaact atcaatataa ccagtacaca gaggaagaaa agaataacat caacttcaac
2340atcgacgact tatcttcaaa gctgaatgaa tctattaaca aagcgatgat taatattaac
2400aagttcttga accaatgtag tgtcagctat ctgatgaact cgatgatccc ttacggtgtg
2460aaacgtctgg aagacttcga tgcaagcctt aaagatgccc ttctgaagta tatttacgat
2520aatcgcggaa ctcttattgg ccaagtggat cgcttaaaag ataaagtcaa caacacgctg
2580agtacagaca tcccttttca gctgtctaaa tatgtggaca atcagcgcct gctgtccacg
2640tttacggaat acatcaaaaa catcatcaac actagtattc tgaacttgcg ttacgagagt
2700aaccatctga ttgatctgag ccgttacgca tctaaaatca acatcggatc caaggtgaac
2760ttcgatccta tcgacaaaaa ccagattcaa ttgttcaact tagaatcgtc aaagattgaa
2820gttatcttaa aaaatgcgat tgtatataat tcaatgtacg aaaatttctc tacgagcttt
2880tggattcgta ttccgaaata tttcaacagt atctctttaa acaacgagta tactatcatc
2940aattgtatgg agaataacag cgggtggaaa gtgagcctta actatggtga aatcatctgg
3000actctgcagg acactcaaga aattaaacaa cgcgtggtgt ttaaatactc acagatgatt
3060aacatctcgg attatattaa tcgctggatt tttgtgacaa ttactaacaa ccggctgaac
3120aacagcaaaa tttacattaa cggtcgcctg atcgatcaga aaccaatcag taatctcggt
3180aacattcacg catcgaataa tatcatgttc aaactggatg gttgtcgcga cacgcaccgt
3240tacatttgga tcaaatactt caatttattc gacaaagaac tcaacgaaaa ggagattaag
3300gatctttatg acaatcagtc taattcgggt attctgaaag acttttgggg tgattacctt
3360cagtacgata aaccgtatta tatgttaaac ttatatgatc cgaataaata cgttgacgtc
3420aacaacgttg gcattcgtgg ctatatgtat ctgaaagggc cgcgtggcag cgtgatgacc
3480actaacattt acttaaactc ctccctctat cgcggtacta aatttattat caagaaatat
3540gcctctggca acaaggacaa tatcgtacgc aataacgatc gcgtctacat taacgtggtg
3600gtgaagaata aagaatatcg tctggcgacc aatgctagtc aggcgggcgt ggagaaaatt
3660ctgtctgcac ttgaaatccc ggatgtgggt aatttatccc aggtggttgt gatgaaaagt
3720aaaaatgacc aagggatcac caataaatgc aaaatgaatc tgcaagataa caacggcaac
3780gacattggtt ttatcggctt ccaccaattc aataatatcg cgaagcttgt ggcctcaaat
3840tggtacaacc gtcagattga gcgcagctcc cgcactttag gctgtagctg ggagttcatt
3900ccggtagatg acggttgggg agaacgccca ttgcaccatc atcaccatca ctgagcggcc
3960gcataatgct taagtcgaac agattgatat gtagctataa gtaatcgtat tgtacacggc
4020cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
4080atcttagtat attagttaag tataagaagg agatatacca tgggcgaatc tctgttcaag
4140ggtccgcgtg attataaccc gatatcttct tctatttgtc atctgactaa cgaaagcgac
4200ggccacacga cttctctgta cggtatcggt ttcggtccgt tcatcattac caacaagcat
4260ctgttccgcc gtaacaacgg taccctgctg gttcaatctc tgcacggcgt cttcaaggta
4320aaagacacca ctacgctgca gcagcacctg gtcgacggcc gtgacatgat catcatccgc
4380atgccgaaag attttccgcc gttcccgcaa aaactgaagt ttcgtgaacc gcaacgcgaa
4440gaacgtattt gcctggttac caccaacttt cagaccaaaa gcatgtcttc tatggtttcc
4500gatacctctt gcaccttccc aagcggtgac ggtattttct ggaaacattg gattcagacc
4560aaagatggtc agtgcggctc tccgctggtg tctacgcgtg acggtttcat cgttggtatc
4620cattctgctt ctaacttcac taacactaac aactacttta cttccgttcc gaaaaacttc
4680atggagctgc tgactaacca agaggcccag cagtgggtgt ccggttggcg cctgaacgca
4740gattctgtac tgtggggtgg tcataaggta ttcatgaaca aaccggagga gccgttccag
4800ccggtcaaag aggcgaccca gctgatgaac gaactggttt actctcagta a
485191732DNAArtificial SequenceTEV variant 7 91atgggcgaat ctctgttcaa
gggtccgcgt gattataacc cgatatcttc ttctatttgt 60catctgacta acgaaagcga
cggccacacg acttctctgt acggtatcgg tttcggtccg 120ttcatcatta ccaacaagca
tctgttccgc cgtaacaacg gtaccctgct ggttcaatct 180ctgcacggcg tcttcaaggt
aaaagacacc actacgctgc agcagcacct ggtcgacggc 240cgtgacatga tcatcatccg
catgccgaaa gattttccgc cgttcccgca aaaactgaag 300tttcgtgaac cgcaacgcga
agaacgtatt tgcctggtta ccaccaactt tcagaccaaa 360agcatgtctt ctatggtttc
cgatacctct tgcaccttcc caagcggtga cggtattttc 420tggaaacatt ggatccagac
caaagatggt cagtgcggct ctccgctggt gtctacgcgt 480gacggtttca tcgttggtat
ccattctgct tctaacttca ctaacactaa caactacttt 540acttccgttc cgaaaaactt
catggagctg ctgactaacc aagaggccca gcagtgggtg 600tccggttggc gcctgaacgc
agattctgta ctgtggggtg gtcataaggt attcatgaac 660aaaccggagg agccgttcca
gccggtcaaa gaggcgaccc agctgatgaa cgaactggtt 720tactctcagt aa
73292415DNAArtificial
SequenceIntervening sequence containing transcriptional and
translational sites and T7 termination site. 92aagcttgtgg cctcaaattg
gtacaaccgt cagattgagc gcagctcccg cactttaggc 60tgtagctggg agttcattcc
ggtagatgac ggttggggag aacgcccatt gcaccatcat 120caccatcact gagcggccgc
ataatgctta agtcgaacag attgatatgt agctataagt 180aattgtatga ctgaacctag
gctgctgcca ccgctgagca ataactagca taaccccttg 240gggcctctaa acgggtcttg
aggggttttt tgctgatcgt atactctcag gcatctatga 300gttgtacacg tccgcataat
cgaaattaat acgactcact ataggggaat tgtgagcgga 360taacaattcc ccatcttagt
atattagtta agtataagaa ggagatatac catgg 415934965DNAArtificial
SequenceBoNT/A-TEV and TEV protease variant 4 open reading frames
with the intervening transcription and translation elements and
termination site. 93atgccgttcg taaacaaaca gttcaactat aaagacccag
tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga
aagcatttaa aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg tcttactatg
atagcacata cctgagtacc 240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac
tgttcgagcg catttattcg 300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg
gcattccgtt ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact
gcatcaatgt gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat gaagtcctga
atctgacgcg gaatggctat 540ggatcgacgc agtatattcg tttttctcca gatttcacat
ttggatttga agaaagcctc 600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg
cgacggaccc agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg
gaatcgccat taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat gcaaaattta
ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta caataaattc aaagacattg
catcaacctt aaacaaggcg 900aaaagcattg tgggtaccac ggctagctta caatatatga
aaaacgtttt caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg
ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat aaagccgtct
ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta tgatggcttt aatctgcgca
atacgaatct ggcggcgaac 1200tttaacggcc agaacaccga aatcaacaac atgaacttta
ctaaactgaa aaattttacc 1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta
ttatcaccag caaaaccaaa 1320tccttgggcg gtggtggcga aaacctgtac ttccagggcg
gtggcggtgg tgataagggc 1380tataacaagg ccttcaatga tttatgcatc aaggtgaaca
actgggactt gtttttctct 1440ccatctgaag ataattttac taacgacttg aacaaaggag
aggaaattac ttccgatacc 1500aacatcgaag cagcggaaga gaatattagt ctagatctta
ttcaacaata ttacctgacc 1560tttaattttg ataacgagcc tgagaacatt tccattgaga
atctcagctc tgacatcatc 1620ggccagctgg aactgatgcc gaatatcgaa cgctttccta
atggaaagaa atatgaattg 1680gacaaataca ccatgttcca ctatctccgc gcgcaggagt
ttgagcacgg caagtctcgt 1740attgctctga ccaattcggt aaacgaagcc cttttaaatc
cttcgcgtgt gtacaccttt 1800ttctcaagcg attatgttaa aaaagtgaac aaggcgaccg
aagcggcgat gtttttggga 1860tgggtggaac aactggtata tgactttacg gatgaaactt
ctgaagtctc gaccaccgac 1920aaaattgccg atattaccat tatcattccc tatattggcc
ctgcactgaa cattggtaac 1980atgctgtata aagatgattt tgtgggcgcc ctgatctttt
caggcgctgt tatcctgctg 2040gaatttatcc cggaaatcgc cattccagta ctcggtacct
ttgcgctggt gtcctatatc 2100gcaaacaaag ttttgactgt ccagacgatc gacaacgcgc
tcagtaaacg taacgaaaaa 2160tgggatgagg tgtataagta tattgttacc aactggctcg
ctaaagtaaa cacccagatt 2220gacctgattc gcaagaagat gaaagaagcg ctggaaaacc
aagcagaagc gaccaaagct 2280attatcaact atcaatataa ccagtacaca gaggaagaaa
agaataacat caacttcaac 2340atcgacgact tatcttcaaa gctgaatgaa tctattaaca
aagcgatgat taatattaac 2400aagttcttga accaatgtag tgtcagctat ctgatgaact
cgatgatccc ttacggtgtg 2460aaacgtctgg aagacttcga tgcaagcctt aaagatgccc
ttctgaagta tatttacgat 2520aatcgcggaa ctcttattgg ccaagtggat cgcttaaaag
ataaagtcaa caacacgctg 2580agtacagaca tcccttttca gctgtctaaa tatgtggaca
atcagcgcct gctgtccacg 2640tttacggaat acatcaaaaa catcatcaac actagtattc
tgaacttgcg ttacgagagt 2700aaccatctga ttgatctgag ccgttacgca tctaaaatca
acatcggatc caaggtgaac 2760ttcgatccta tcgacaaaaa ccagattcaa ttgttcaact
tagaatcgtc aaagattgaa 2820gttatcttaa aaaatgcgat tgtatataat tcaatgtacg
aaaatttctc tacgagcttt 2880tggattcgta ttccgaaata tttcaacagt atctctttaa
acaacgagta tactatcatc 2940aattgtatgg agaataacag cgggtggaaa gtgagcctta
actatggtga aatcatctgg 3000actctgcagg acactcaaga aattaaacaa cgcgtggtgt
ttaaatactc acagatgatt 3060aacatctcgg attatattaa tcgctggatt tttgtgacaa
ttactaacaa ccggctgaac 3120aacagcaaaa tttacattaa cggtcgcctg atcgatcaga
aaccaatcag taatctcggt 3180aacattcacg catcgaataa tatcatgttc aaactggatg
gttgtcgcga cacgcaccgt 3240tacatttgga tcaaatactt caatttattc gacaaagaac
tcaacgaaaa ggagattaag 3300gatctttatg acaatcagtc taattcgggt attctgaaag
acttttgggg tgattacctt 3360cagtacgata aaccgtatta tatgttaaac ttatatgatc
cgaataaata cgttgacgtc 3420aacaacgttg gcattcgtgg ctatatgtat ctgaaagggc
cgcgtggcag cgtgatgacc 3480actaacattt acttaaactc ctccctctat cgcggtacta
aatttattat caagaaatat 3540gcctctggca acaaggacaa tatcgtacgc aataacgatc
gcgtctacat taacgtggtg 3600gtgaagaata aagaatatcg tctggcgacc aatgctagtc
aggcgggcgt ggagaaaatt 3660ctgtctgcac ttgaaatccc ggatgtgggt aatttatccc
aggtggttgt gatgaaaagt 3720aaaaatgacc aagggatcac caataaatgc aaaatgaatc
tgcaagataa caacggcaac 3780gacattggtt ttatcggctt ccaccaattc aataatatcg
cgaagcttgt ggcctcaaat 3840tggtacaacc gtcagattga gcgcagctcc cgcactttag
gctgtagctg ggagttcatt 3900ccggtagatg acggttgggg agaacgccca ttgcaccatc
atcaccatca ctgagcggcc 3960gcataatgct taagtcgaac agattgatat gtagctataa
gtaattgtat gactgaacct 4020aggctgctgc caccgctgag caataactag cataacccct
tggggcctct aaacgggtct 4080tgaggggttt tttgctgatc gtatactctc aggcatctat
gagttgtaca cgtccgcata 4140atcgaaatta atacgactca ctatagggga attgtgagcg
gataacaatt ccccatctta 4200gtatattagt taagtataag aaggagatat accatgggcg
aatctctgtt caagggtccg 4260cgtgattata acccgatatc ttcttctatt tgtcatctga
ctaacgaaag cgacggccac 4320acgacttctc tgtacggtat cggtttcggt ccgttcatca
ttaccaacaa gcatctgttc 4380cgccgtaaca acggtaccct gctggttcaa tctctgcacg
gcgtcttcaa ggtaaaagac 4440accactacgc tgcagcagca cctggtcgac ggccgtgaca
tgatcatcat ccgcatgccg 4500aaagattttc cgccgttccc gcaaaaactg aagtttcgtg
aaccgcaacg cgaagaacgt 4560atttgcctgg ttaccaccaa ctttcagacc aaaagcatgt
cttctatggt ttccgatacc 4620tcttgcacct tcccaagcgg tgacggtatt ttctggaaac
attggattca gaccaaagat 4680ggtcagtgcg gctctccgct ggtgtctacg cgtgacggtt
tcatcgttgg tatccattct 4740gcttctaact tcactaacac taacaactac tttacttccg
ttccgaaaaa cttcatggag 4800ctgctgacta accaagaggc ccagcagtgg gtgtccggtt
ggcgcctgaa cgcagattct 4860gtactgtggg gtggtcataa ggtattcatg aacaaaccgg
aggagccgtt ccagccggtc 4920aaagaggcga cccagctgat gaacgaactg gtttactctc
agtaa 4965942697DNAArtificial SequenceOpen reading
frame for NociLHN/A-TEV 94atgccgttcg taaacaaaca gttcaactat aaagacccag
tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga
aagcatttaa aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg tcttactatg
atagcacata cctgagtacc 240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac
tgttcgagcg catttattcg 300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg
gcattccgtt ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact
gcatcaatgt gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat gaagtcctga
atctgacgcg gaatggctat 540ggatcgacgc agtatattcg tttttctcca gatttcacat
ttggatttga agaaagcctc 600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg
cgacggaccc agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg
gaatcgccat taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat gcaaaattta
ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta caataaattc aaagacattg
catcaacctt aaacaaggcg 900aaaagcattg tgggtaccac ggctagctta caatatatga
aaaacgtttt caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg
ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat aaagccgtct
ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta tgatggcttt aatctgcgca
atacgaatct ggcggcgaac 1200tttaacggcc agaacaccga aatcaacaac atgaacttta
ctaaactgaa aaattttacc 1260ggcttgtttg aattctataa gctcctgtgt gtccgcggta
ttatcaccag caaagaaaac 1320ctgtacttcc agttcggtgg ttttaccggc gctcgtaaat
ctgcacgtaa acgcaagaat 1380caggctctgg ctggtggcgg tggctctggt ggtggcggta
gcggcggtgg cggttctgcg 1440ctcaatgatt tatgcatcaa ggtgaacaac tgggacttgt
ttttctctcc atctgaagat 1500aattttacta acgacttgaa caaaggagag gaaattactt
ccgataccaa catcgaagca 1560gcggaagaga atattagtct agatcttatt caacaatatt
acctgacctt taattttgat 1620aacgagcctg agaacatttc cattgagaat ctcagctctg
acatcatcgg ccagctggaa 1680ctgatgccga atatcgaacg ctttcctaat ggaaagaaat
atgaattgga caaatacacc 1740atgttccact atctccgcgc gcaggagttt gagcacggca
agtctcgtat tgctctgacc 1800aattcggtaa acgaagccct tttaaatcct tcgcgtgtgt
acaccttttt ctcaagcgat 1860tatgttaaaa aagtgaacaa ggcgaccgaa gcggcgatgt
ttttgggatg ggtggaacaa 1920ctggtatatg actttacgga tgaaacttct gaagtctcga
ccaccgacaa aattgccgat 1980attaccatta tcattcccta tattggccct gcactgaaca
ttggtaacat gctgtataaa 2040gatgattttg tgggcgccct gatcttttca ggcgctgtta
tcctgctgga atttatcccg 2100gaaatcgcca ttccagtact cggtaccttt gcgctggtgt
cctatatcgc aaacaaagtt 2160ttgactgtcc agacgatcga caacgcgctc agtaaacgta
acgaaaaatg ggatgaggtg 2220tataagtata ttgttaccaa ctggctcgct aaagtaaaca
cccagattga cctgattcgc 2280aagaagatga aagaagcgct ggaaaaccaa gcagaagcga
ccaaagctat tatcaactat 2340caatataacc agtacacaga ggaagaaaag aataacatca
acttcaacat cgacgactta 2400tcttcaaagc tgaatgaatc tattaacaaa gcgatgatta
atattaacaa gttcttgaac 2460caatgtagtg tcagctatct gatgaactcg atgatccctt
acggtgtgaa acgtctggaa 2520gacttcgatg caagccttaa agatgccctt ctgaagtata
tttacgataa tcgcggaact 2580cttattggcc aagtggatcg cttaaaagat aaagtcaaca
acacgctgag tacagacatc 2640ccttttcagc tgtctaaata tgtggacaat cagcgccacc
atcaccatca ccactaa 269795898PRTArtificial SequenceNociLHN/A-TEV
95Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1
5 10 15 Val Asp Ile Ala
Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20
25 30 Val Lys Ala Phe Lys Ile His Asn Lys
Ile Trp Val Ile Pro Glu Arg 35 40
45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro
Pro Glu 50 55 60
Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65
70 75 80 Asp Asn Glu Lys Asp
Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85
90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met
Leu Leu Thr Ser Ile Val 100 105
110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu
Lys 115 120 125 Val
Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130
135 140 Arg Ser Glu Glu Leu Asn
Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150
155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu
Val Leu Asn Leu Thr 165 170
175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe
180 185 190 Thr Phe Gly
Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195
200 205 Gly Ala Gly Lys Phe Ala Thr Asp
Pro Ala Val Thr Leu Ala His Glu 210 215
220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn Pro Asn225 230 235
240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu
245 250 255 Glu Val Ser Phe
Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260
265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu
Phe Arg Leu Tyr Tyr Tyr Asn 275 280
285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser
Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305
310 315 320 Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325
330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr Glu Asp 340 345
350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu
Asn 355 360 365 Phe
Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370
375 380 Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390
395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met
Asn Phe Thr Lys Leu 405 410
415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg
420 425 430 Gly Ile Ile
Thr Ser Lys Glu Asn Leu Tyr Phe Gln Phe Gly Gly Phe 435
440 445 Thr Gly Ala Arg Lys Ser Ala Arg
Lys Arg Lys Asn Gln Ala Leu Ala 450 455
460 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ala465 470 475
480 Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser
485 490 495 Pro Ser Glu Asp
Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile 500
505 510 Thr Ser Asp Thr Asn Ile Glu Ala Ala
Glu Glu Asn Ile Ser Leu Asp 515 520
525 Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu
Pro Glu 530 535 540
Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu545
550 555 560 Leu Met Pro Asn Ile
Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu 565
570 575 Asp Lys Tyr Thr Met Phe His Tyr Leu Arg
Ala Gln Glu Phe Glu His 580 585
590 Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu
Leu 595 600 605 Asn
Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys 610
615 620 Val Asn Lys Ala Thr Glu
Ala Ala Met Phe Leu Gly Trp Val Glu Gln625 630
635 640 Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu
Val Ser Thr Thr Asp 645 650
655 Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu
660 665 670 Asn Ile Gly
Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile 675
680 685 Phe Ser Gly Ala Val Ile Leu Leu
Glu Phe Ile Pro Glu Ile Ala Ile 690 695
700 Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala
Asn Lys Val705 710 715
720 Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys
725 730 735 Trp Asp Glu Val
Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys Val 740
745 750 Asn Thr Gln Ile Asp Leu Ile Arg Lys
Lys Met Lys Glu Ala Leu Glu 755 760
765 Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr
Asn Gln 770 775 780
Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu785
790 795 800 Ser Ser Lys Leu Asn
Glu Ser Ile Asn Lys Ala Met Ile Asn Ile Asn 805
810 815 Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr
Leu Met Asn Ser Met Ile 820 825
830 Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys
Asp 835 840 845 Ala
Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly Gln 850
855 860 Val Asp Arg Leu Lys Asp
Lys Val Asn Asn Thr Leu Ser Thr Asp Ile865 870
875 880 Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln
Arg His His His His 885 890
895 His His962709DNAArtificial SequenceOpen reading frame for
DynLHN/A-TEV 96atgccgttcg taaacaaaca gttcaactat aaagacccag tcaacggcgt
ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga aagcatttaa
aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc cggaagaagg
agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg tcttactatg atagcacata
cctgagtacc 240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac tgttcgagcg
catttattcg 300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg gcattccgtt
ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact gcatcaatgt
gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca ttggcccgag
cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat gaagtcctga atctgacgcg
gaatggctat 540ggatcgacgc agtatattcg tttttctcca gatttcacat ttggatttga
agaaagcctc 600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg cgacggaccc
agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg gaatcgccat
taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt cgggcttaga
agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat gcaaaattta ttgatagtct
gcaagaaaac 840gaatttcggc tgtactatta caataaattc aaagacattg catcaacctt
aaacaaggcg 900aaaagcattg tgggtaccac ggctagctta caatatatga aaaacgtttt
caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg ataaactgaa
atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact ttgtcaaatt
cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat aaagccgtct ttaagatcaa
catcgtaccg 1140aaagttaact acaccatcta tgatggcttt aatctgcgca atacgaatct
ggcggcgaac 1200tttaacggcc agaacaccga aatcaacaac atgaacttta ctaaactgaa
aaattttacc 1260ggcttgtttg aattctataa gctcctgtgt gtccgtggta ttatcaccag
caaagaaaac 1320ctgtacttcc agtatggcgg tttcctgcgt cgcattcgtc ctaagcttaa
atgggataac 1380caggctcttg ctggtggtgg tggctctggt ggtggcggta gcggcggtgg
tggttctgca 1440ctcaatgatt tatgtatcaa ggtgaacaac tgggacttgt ttttctctcc
atctgaagat 1500aattttacta acgacttgaa caaaggagag gaaattactt ccgataccaa
catcgaagca 1560gcggaagaga atattagtct agatcttatt caacaatatt acctgacctt
taattttgat 1620aacgagcctg agaacatttc cattgagaat ctcagctctg acatcatcgg
ccagctggaa 1680ctgatgccga atatcgaacg ctttcctaat ggaaagaaat atgaattgga
caaatacacc 1740atgttccact atctccgcgc gcaggagttt gagcacggca agtctcgtat
tgctctgacc 1800aattcggtaa acgaagccct tttaaatcct tcgcgtgtgt acaccttttt
ctcaagcgat 1860tatgttaaaa aagtgaacaa ggcgaccgaa gcggcgatgt ttttgggatg
ggtggaacaa 1920ctggtatatg actttacgga tgaaacttct gaagtctcga ccaccgacaa
aattgccgat 1980attaccatta tcattcccta tattggccct gcactgaaca ttggtaacat
gctgtataaa 2040gatgattttg tgggcgccct gatcttttca ggcgctgtta tcctgctgga
atttatcccg 2100gaaatcgcca ttccagtact cggtaccttt gcgctggtgt cctatatcgc
aaacaaagtt 2160ttgactgtcc agacgatcga caacgcgctc agtaaacgta acgaaaaatg
ggatgaggtg 2220tataagtata ttgttaccaa ctggctcgct aaagtaaaca cccagattga
cctgattcgc 2280aagaagatga aagaagcgct ggaaaaccaa gcagaagcga ccaaagctat
tatcaactat 2340caatataacc agtacacaga ggaagaaaag aataacatca acttcaacat
cgacgactta 2400tcttcaaagc tgaatgaatc tattaacaaa gcgatgatta atattaacaa
gttcttgaac 2460caatgtagtg tcagctatct gatgaactcg atgatccctt acggtgtgaa
acgtctggaa 2520gacttcgatg caagccttaa agatgccctt ctgaagtata tttacgataa
tcgcggaact 2580cttattggcc aagtggatcg cttaaaagat aaagtcaaca acacgctgag
tacagacatc 2640ccttttcagc tgtctaaata tgtggacaat cagcgcctgc tgtccacgca
ccatcaccat 2700caccactaa
270997902PRTArtificial SequenceDynLHN/A-TEV 97Met Pro Phe Val
Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5
10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro
Asn Ala Gly Gln Met Gln Pro 20 25
30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro
Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50
55 60 Ala Lys Gln Val Pro
Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70
75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly
Val Thr Lys Leu Phe Glu 85 90
95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile
Val 100 105 110 Arg
Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115
120 125 Val Ile Asp Thr Asn Cys
Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135
140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly
Pro Ser Ala Asp Ile145 150 155
160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr
165 170 175 Arg Asn Gly
Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180
185 190 Thr Phe Gly Phe Glu Glu Ser Leu
Glu Val Asp Thr Asn Pro Leu Leu 195 200
205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu
Ala His Glu 210 215 220
Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225
230 235 240 Arg Val Phe Lys Val
Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245
250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe
Gly Gly His Asp Ala Lys 260 265
270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr
Asn 275 280 285 Lys
Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290
295 300 Gly Thr Thr Ala Ser Leu
Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310
315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe
Ser Val Asp Lys Leu 325 330
335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp
340 345 350 Asn Phe Val
Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355
360 365 Phe Asp Lys Ala Val Phe Lys Ile
Asn Ile Val Pro Lys Val Asn Tyr 370 375
380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu
Ala Ala Asn385 390 395
400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu
405 410 415 Lys Asn Phe Thr
Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420
425 430 Gly Ile Ile Thr Ser Lys Glu Asn Leu
Tyr Phe Gln Tyr Gly Gly Phe 435 440
445 Leu Arg Arg Ile Arg Pro Lys Leu Lys Trp Asp Asn Gln Ala
Leu Ala 450 455 460
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala465
470 475 480 Leu Asn Asp Leu Cys
Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser 485
490 495 Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu
Asn Lys Gly Glu Glu Ile 500 505
510 Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu
Asp 515 520 525 Leu
Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu 530
535 540 Asn Ile Ser Ile Glu Asn
Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu545 550
555 560 Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly
Lys Lys Tyr Glu Leu 565 570
575 Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His
580 585 590 Gly Lys Ser
Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu 595
600 605 Asn Pro Ser Arg Val Tyr Thr Phe
Phe Ser Ser Asp Tyr Val Lys Lys 610 615
620 Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp
Val Glu Gln625 630 635
640 Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp
645 650 655 Lys Ile Ala Asp
Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu 660
665 670 Asn Ile Gly Asn Met Leu Tyr Lys Asp
Asp Phe Val Gly Ala Leu Ile 675 680
685 Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile
Ala Ile 690 695 700
Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys Val705
710 715 720 Leu Thr Val Gln Thr
Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu Lys 725
730 735 Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr
Asn Trp Leu Ala Lys Val 740 745
750 Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu
Glu 755 760 765 Asn
Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn Gln 770
775 780 Tyr Thr Glu Glu Glu Lys
Asn Asn Ile Asn Phe Asn Ile Asp Asp Leu785 790
795 800 Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala
Met Ile Asn Ile Asn 805 810
815 Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met Ile
820 825 830 Pro Tyr Gly
Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys Asp 835
840 845 Ala Leu Leu Lys Tyr Ile Tyr Asp
Asn Arg Gly Thr Leu Ile Gly Gln 850 855
860 Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser
Thr Asp Ile865 870 875
880 Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser Thr
885 890 895 His His His His
His His 900 98320DNAArtificial SequenceDNA fragment
encoding a di-chain loop region comprising an integrated TEV
protease cleavage site-Galanin binding domain 98gaattctaca
agctgctgtg cgtggacggc atcattacct ccaaaactaa atctgaaaac 60ctgtacttcc
agggctggac tttgaactct gctggttatc tcctgggccc acatgcggtt 120gctcttgctg
gtggcggtgg ctctggcggt ggcggtagcg gcggtggcgg ttctgcacta 180gtgcttcagt
gtatcaaggt taacaactgg gatttattct tcagcccgag tgaagacaac 240ttcaccaacg
acctgaacaa aggtgaagaa atcacctcag atactaacat cgaagcagcc 300gaagaaaaca
tcagtctaga
32099106PRTArtificial SequenceDi-chain loop region comprising an
integrated TEV protease cleavage site-Galanin binding domain 99Glu
Phe Tyr Lys Leu Leu Cys Val Asp Gly Ile Ile Thr Ser Lys Thr1
5 10 15 Lys Ser Glu Asn Leu Tyr
Phe Gln Gly Trp Thr Leu Asn Ser Ala Gly 20 25
30 Tyr Leu Leu Gly Pro His Ala Val Ala Leu Ala
Gly Gly Gly Gly Ser 35 40 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Leu Gln Cys
50 55 60 Ile Lys Val
Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn65 70
75 80 Phe Thr Asn Asp Leu Asn Lys Gly
Glu Glu Ile Thr Ser Asp Thr Asn 85 90
95 Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu
100 105 1002703DNAArtificial SequenceOpen reading
frame for GalLHN/A-TEV 100atgccgttcg taaacaaaca gttcaactat aaagacccag
tcaacggcgt ggacattgcc 60tatatcaaaa tcccgaatgc gggtcaaatg cagcccgtga
aagcatttaa aatccataac 120aaaatttggg tgatcccgga gcgcgatacg ttcacgaacc
cggaagaagg agatttaaac 180ccaccgcctg aggctaaaca ggtcccggtg tcttactatg
atagcacata cctgagtacc 240gacaatgaaa aggacaacta cctgaaaggt gttaccaaac
tgttcgagcg catttattcg 300acagatctcg gtcgcatgtt gctgacttct attgtgcgcg
gcattccgtt ttggggtggt 360agcaccatcg atacagaact caaagtgatt gacaccaact
gcatcaatgt gattcagcct 420gatgggagct accggtccga agagcttaac ctcgtaatca
ttggcccgag cgcggatatt 480atccaattcg aatgtaaatc ttttgggcat gaagtcctga
atctgacgcg gaatggctat 540ggatcgacgc agtatattcg tttttctcca gatttcacat
ttggatttga agaaagcctc 600gaagttgata cgaaccctct tttaggcgcg ggaaaattcg
cgacggaccc agcggtgacc 660ttggcacatg aacttattca tgccgggcat cgcttgtatg
gaatcgccat taacccgaac 720cgtgttttca aggtgaatac gaacgcgtat tacgagatgt
cgggcttaga agtgtccttt 780gaagaactgc gcacgtttgg cggtcatgat gcaaaattta
ttgatagtct gcaagaaaac 840gaatttcggc tgtactatta caataaattc aaagacattg
catcaacctt aaacaaggcg 900aaaagcattg tgggtaccac ggctagctta caatatatga
aaaacgtttt caaagaaaaa 960tacctcctta gcgaagacac ttccggcaaa ttctctgtcg
ataaactgaa atttgataaa 1020ctgtataaaa tgctcaccga gatctacaca gaggataact
ttgtcaaatt cttcaaggtc 1080ttgaatcgga aaacctatct gaacttcgat aaagccgtct
ttaagatcaa catcgtaccg 1140aaagttaact acaccatcta tgatggcttt aatctgcgca
atacgaatct ggcggcgaac 1200tttaacggcc agaacaccga aatcaacaac atgaacttta
ctaaactgaa aaattttacc 1260ggcttgtttg aattctacaa gctgctgtgc gtggacggca
tcattacctc caaaactaaa 1320tctgaaaacc tgtacttcca gggctggact ttgaactctg
ctggttatct cctgggccca 1380catgcggttg ctcttgctgg tggcggtggc tctggcggtg
gcggtagcgg cggtggcggt 1440tctgcactag tgcttcagtg tatcaaggtt aacaactggg
atttattctt cagcccgagt 1500gaagacaact tcaccaacga cctgaacaaa ggtgaagaaa
tcacctcaga tactaacatc 1560gaagcagccg aagaaaacat cagtctagat cttattcaac
aatattacct gacctttaat 1620tttgataacg agcctgagaa catttccatt gagaatctca
gctctgacat catcggccag 1680ctggaactga tgccgaatat cgaacgcttt cctaatggaa
agaaatatga attggacaaa 1740tacaccatgt tccactatct ccgcgcgcag gagtttgagc
acggcaagtc tcgtattgct 1800ctgaccaatt cggtaaacga agccctttta aatccttcgc
gtgtgtacac ctttttctca 1860agcgattatg ttaaaaaagt gaacaaggcg accgaagcgg
cgatgttttt gggatgggtg 1920gaacaactgg tatatgactt tacggatgaa acttctgaag
tctcgaccac cgacaaaatt 1980gccgatatta ccattatcat tccctatatt ggccctgcac
tgaacattgg taacatgctg 2040tataaagatg attttgtggg cgccctgatc ttttcaggcg
ctgttatcct gctggaattt 2100atcccggaaa tcgccattcc agtactcggt acctttgcgc
tggtgtccta tatcgcaaac 2160aaagttttga ctgtccagac gatcgacaac gcgctcagta
aacgtaacga aaaatgggat 2220gaggtgtata agtatattgt taccaactgg ctcgctaaag
taaacaccca gattgacctg 2280attcgcaaga agatgaaaga agcgctggaa aaccaagcag
aagcgaccaa agctattatc 2340aactatcaat ataaccagta cacagaggaa gaaaagaata
acatcaactt caacatcgac 2400gacttatctt caaagctgaa tgaatctatt aacaaagcga
tgattaatat taacaagttc 2460ttgaaccaat gtagtgtcag ctatctgatg aactcgatga
tcccttacgg tgtgaaacgt 2520ctggaagact tcgatgcaag ccttaaagat gcccttctga
agtatattta cgataatcgc 2580ggaactctta ttggccaagt ggatcgctta aaagataaag
tcaacaacac gctgagtaca 2640gacatccctt ttcagctgtc taaatatgtg gacaatcagc
gccaccatca ccatcaccac 2700taa
2703101900PRTArtificial SequenceGalLHN/A-TEV 101Met
Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1
5 10 15 Val Asp Ile Ala Tyr Ile
Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25
30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp
Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu
50 55 60 Ala Lys Gln
Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70
75 80 Asp Asn Glu Lys Asp Asn Tyr Leu
Lys Gly Val Thr Lys Leu Phe Glu 85 90
95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr
Ser Ile Val 100 105 110
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys
115 120 125 Val Ile Asp Thr
Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130
135 140 Arg Ser Glu Glu Leu Asn Leu Val
Ile Ile Gly Pro Ser Ala Asp Ile145 150
155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val
Leu Asn Leu Thr 165 170
175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe
180 185 190 Thr Phe Gly
Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195
200 205 Gly Ala Gly Lys Phe Ala Thr Asp
Pro Ala Val Thr Leu Ala His Glu 210 215
220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile
Asn Pro Asn225 230 235
240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu
245 250 255 Glu Val Ser Phe
Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260
265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu
Phe Arg Leu Tyr Tyr Tyr Asn 275 280
285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser
Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305
310 315 320 Tyr Leu Leu Ser Glu
Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325
330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr
Glu Ile Tyr Thr Glu Asp 340 345
350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu
Asn 355 360 365 Phe
Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370
375 380 Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390
395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met
Asn Phe Thr Lys Leu 405 410
415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Asp
420 425 430 Gly Ile Ile
Thr Ser Lys Thr Lys Ser Glu Asn Leu Tyr Phe Gln Gly 435
440 445 Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro His Ala Val Ala 450 455
460 Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly465 470 475
480 Ser Ala Leu Val Leu Gln Cys Ile Lys Val Asn Asn Trp Asp Leu Phe
485 490 495 Phe Ser Pro Ser
Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu 500
505 510 Glu Ile Thr Ser Asp Thr Asn Ile Glu
Ala Ala Glu Glu Asn Ile Ser 515 520
525 Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp
Asn Glu 530 535 540
Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln545
550 555 560 Leu Glu Leu Met Pro
Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr 565
570 575 Glu Leu Asp Lys Tyr Thr Met Phe His Tyr
Leu Arg Ala Gln Glu Phe 580 585
590 Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu
Ala 595 600 605 Leu
Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val 610
615 620 Lys Lys Val Asn Lys Ala
Thr Glu Ala Ala Met Phe Leu Gly Trp Val625 630
635 640 Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr
Ser Glu Val Ser Thr 645 650
655 Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro
660 665 670 Ala Leu Asn
Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala 675
680 685 Leu Ile Phe Ser Gly Ala Val Ile
Leu Leu Glu Phe Ile Pro Glu Ile 690 695
700 Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr
Ile Ala Asn705 710 715
720 Lys Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn
725 730 735 Glu Lys Trp Asp
Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala 740
745 750 Lys Val Asn Thr Gln Ile Asp Leu Ile
Arg Lys Lys Met Lys Glu Ala 755 760
765 Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr
Gln Tyr 770 775 780
Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp785
790 795 800 Asp Leu Ser Ser Lys
Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn 805
810 815 Ile Asn Lys Phe Leu Asn Gln Cys Ser Val
Ser Tyr Leu Met Asn Ser 820 825
830 Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser
Leu 835 840 845 Lys
Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile 850
855 860 Gly Gln Val Asp Arg Leu
Lys Asp Lys Val Asn Asn Thr Leu Ser Thr865 870
875 880 Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp
Asn Gln Arg His His 885 890
895 His His His His 900 102314DNAArtificial SequenceDNA
fragment encoding a di-chain loop region comprising an integrated
TEV protease cleavage site-Nociceptin binding domain 102gaattctata
agctcctgtg tgtccgcggt attatcacca gcaaagaaaa cctgtacttc 60cagttcggtg
gttttaccgg cgctcgtaaa tctgcacgta aacgcaagaa tcaggctctg 120gctggtggcg
gtggctctgg tggtggcggt agcggcggtg gcggttctgc gctcaatgat 180ttatgcatca
aggtgaacaa ctgggacttg tttttctctc catctgaaga taattttact 240aacgacttga
acaaaggaga ggaaattact tccgatacca acatcgaagc agcggaagag 300aatattagtc
taga
314103104PRTArtificial SequenceDi-chain loop region comprising an
integrated TEV protease cleavage site-Nociceptin binding domain
103Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile Thr Ser Lys Glu1
5 10 15 Asn Leu Tyr Phe
Gln Phe Gly Gly Phe Thr Gly Ala Arg Lys Ser Ala 20
25 30 Arg Lys Arg Lys Asn Gln Ala Leu Ala
Gly Gly Gly Gly Ser Gly Gly 35 40
45 Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Asn Asp Leu Cys
Ile Lys 50 55 60
Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn Phe Thr65
70 75 80 Asn Asp Leu Asn Lys
Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu 85
90 95 Ala Ala Glu Glu Asn Ile Ser Leu
100 104314DNAArtificial SequenceDNA fragment encoding a
di-chain loop region comprising an integrated TEV protease cleavage
site-Dynorphin binding domain 104gaattctata agctcctgtg tgtccgtggt
attatcacca gcaaagaaaa cctgtacttc 60cagtatggcg gtttcctgcg tcgcattcgt
cctaagctta aatgggataa ccaggctctt 120gctggtggtg gtggctctgg tggtggcggt
agcggcggtg gtggttctgc actcaatgat 180ttatgtatca aggtgaacaa ctgggacttg
tttttctctc catctgaaga taattttact 240aacgacttga acaaaggaga ggaaattact
tccgatacca acatcgaagc agcggaagag 300aatattagtc taga
314105104PRTArtificial SequenceDi-chain
loop region comprising an integrated TEV protease cleavage
site-Dynorphin binding domain 105Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly
Ile Ile Thr Ser Lys Glu1 5 10
15 Asn Leu Tyr Phe Gln Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro
Lys 20 25 30 Leu
Lys Trp Asp Asn Gln Ala Leu Ala Gly Gly Gly Gly Ser Gly Gly 35
40 45 Gly Gly Ser Gly Gly Gly
Gly Ser Ala Leu Asn Asp Leu Cys Ile Lys 50 55
60 Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser
Glu Asp Asn Phe Thr65 70 75
80 Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn Ile Glu
85 90 95 Ala Ala Glu
Glu Asn Ile Ser Leu 100 106750DNAArtificial
SequenceOpen reading frame encoding TEV protease variant 7
106ccatggatgg gtggcgaatc tctgttcaag ggtccgcgtg attataaccc gatatcttct
60actatttgtc atctgactaa cgaaagcgac ggccacacga cttctctgta cggtatcggt
120ttcggtccgt tcatcattac caacaagcat ctgttccgcc gtaacaacgg taccctgctg
180gttcaatctc tgcacggcgt cttcaaggta aaagacacca ctacgctgca gcagcacctg
240gtcgacggcc gtgacatgat catcatccgc atgccgaaag attttccgcc gttcccgcaa
300aaactgaagt ttcgtgaacc gcaacgcgaa gaacgtattt gcctggttac caccaacttt
360cagaccaaaa gcatgtcttc tatggtttcc gatacctctt gcaccttccc aagcggtgac
420ggtattttct ggaaacattg gatccagacc aaagatggtc agtgcggctc tccgctggtg
480tctacgcgtg acggtttcat cgttggtatc cattctgctt ctaacttcac taacactaac
540aactacttta cttccgttcc gaaaaacttc atggagctgc tgactaacca agaggcccag
600cagtgggtgt ccggttggcg cctgaacgca gattctgtac tgtggggtgg tcataaggta
660ttcatgaaca aaccggagga gccgttccag ccggtcaaag aggcgaccca gctgatgaac
720gaactggttt actctcagta atgaaagctt
7501073791DNAArtificial SequenceORF encoding p10-TEV variant 7 and
polH-DynLHn/A-TEV 107agatcttatg cggccgcact cgagtcatta gtggtgatgg
tgatggtggg ttgacagaag 60tctctgatta tcaacatact tcgacagttg gaacgggatg
tctgttgaca gagtgttgtt 120taccttatct ttcagacggt caacttggcc aatgagcgtt
cccctgttat cgtagatgta 180cttaagaagg gcgtctttca gcgaggcgtc gaagtcctcc
agtctcttta caccatatgg 240gatcattgag ttcatcaagt atgatacact gcattggttg
aggaacttgt taatgtttat 300cattgccttg tttatgctct cgttgagttt actagagagg
tcgtcaatgt tgaagttgat 360gttgttcttt tcctcctcgg tgtactggtt gtactgatag
ttaatgatgg cctttgtcgc 420ttcagcctgg ttctccagcg cctccttcat ctttttcctg
atgaggtcga tttgggtgtt 480gaccttagcg agccagttag tcacgatgta tttgtacact
tcatcccact tctcgtttct 540ttttgacaga gcattatcga ttgtttggac agtgaggacc
ttgttagcaa tgtagctgac 600caaagcgaag gtaccaagaa caggaatagc gatctctggg
atgaactcca acaaaatcac 660tgctcccgag aaaatcaacg caccgacgaa gtcgtccttg
tacagcatat tgccgatgtt 720aagagcaggt ccaatgtagg gtatgatgat agtgatgtct
gcgatcttgt ccgtagtcga 780aacttcacta gtctcgtcgg tgaaatcgta aaccaactgt
tcaacccaac ccagaaacat 840cgctgcttcg gttgccttat tcaccttctt aacgtaatcc
gaactgaaga aggtgtagac 900acgagaagga ttgagaagag cctcgttgac cgagttagtg
agggcgattc tactttttcc 960gtgctcaaac tcttgagctc tgaggtagtg gaacatcgtg
tatttgtcca attcgtactt 1020cttgccgtta gggaatctct cgatattggg catgagttcc
agctgtccga tgatgtcgct 1080gctcagattt tcgatagaaa tgttttccgg ctcgttatcg
aaattgaacg tgagatagta 1140ctgctgaatc aggtctagac taatattctc ttccgctgct
tcgatgttgg tatcggaagt 1200aatttcctct cctttgttca agtcgttagt aaaattatct
tcagatggag agaaaaacaa 1260gtcccagttg ttcaccttga tacataaatc attgagtgca
gaaccaccac cgccgctacc 1320gccaccacca gagccaccac caccagcaag agcctggtta
tcccatttaa gcttaggacg 1380aatgcgacgc aggaaaccgc catactggaa gtacaggttt
tctttgctgg tgataatacc 1440acggacacac aggagcttat agaattcaaa caggccggtg
aaattcttga gctttgtgaa 1500gttcatgtta ttgatctcgg tattctgacc attgaagtta
gccgccaaat tggtgttcct 1560aaggttaaag ccatcataga tggtgtagtt cacctttggc
acgatattga tcttaaacac 1620agctttgtcg aagttaagat aagtcttgcg gttcaatacc
ttgaagaact taacaaagtt 1680gtcctcggta tagatctctg taagcatttt gtacagcttg
tcaaacttga gtttgtccac 1740ggaaaacttt ccggaggtgt cctcggaaag caagtacttt
tccttaaaga cgttcttcat 1800atactgaagg ctagccgtgg tgccgactat acttttagcc
ttattcagcg tactggcaat 1860atctttgaat ttgttgtagt aatacagtct gaactcattc
tcttgcaagg agtcgatgaa 1920cttagcatcg tgtccaccga aggtacgaag ttcttcgaag
gagacttcca gaccggacat 1980ctcatagtat gcgttggtgt tcaccttgaa aacgcggttt
ggattgatgg caattccgta 2040cagtctatgg cctgcgtgaa tcagctcgtg agccaaggtc
accgcgggat ctgtggcgaa 2100cttgccagcg cccaacaacg gattagtgtc aacctccaat
gactcttcga agccgaaagt 2160gaaatcgggg gaaaacctga tgtattgagt agaaccataa
ccgtttctgg tcaggttcag 2220cacctcatgg ccgaaggact tacattcaaa ctgaatgatg
tcggcagagg gaccgatgat 2280caccaagttg agttcctctg aacggtagga gccgtcaggt
tggatcacgt tgatacagtt 2340tgtatcgatc actttcagct ctgtatctat ggttgatccg
ccccaaaagg ggattccacg 2400gacgatggaa gtgagcagca tgcgaccgag gtcagtggaa
tagatacgct cgaaaagttt 2460ggtcactccc ttgaggtaat tgtctttctc gttatctgtc
gacaagtacg tggagtcata 2520gtaggacacc ggcacctgct tggcctctgg tggcggattc
aaatctcctt cttcggggtt 2580agtgaaggtg tctctttcgg gaatgaccca tatcttgtta
tgaatcttga aggccttaac 2640aggctgcatt tgaccggcat tcggaatctt gatatacgca
atatcgactc cgttgacagg 2700gtccttatag ttgaattgct tgttgacaaa tcccatggga
ttatatttat aggttttttt 2760attacaaaac tgttacgaaa acagtaaaat acttatttat
ttgcgagatg gttatcattt 2820taattatctc catgatccaa taacctagaa taaaggccga
cctttaattc aacccaacac 2880aatatattat agttaaataa gaattattat caaatcattt
gtatattaat taaaatacta 2940tactgtaaat tacattttat ttacaatcac agatccatat
gggcgagtca ttgttcaagg 3000gaccgagaga ttacaacccc atctcgtcgt caatctgcca
cttgacaaac gaatccgacg 3060gtcacactac ttctctgtac ggtatcggct tcggaccttt
catcatcacc aacaagcatt 3120tgtttaggag aaacaacggt acactccttg tccagtccct
gcacggcgta ttcaaagtca 3180aagataccac gactctgcaa cagcatctgg tcgacggaag
ggacatgata atcattcgca 3240ttcctaaaga cttcccaccc ttccctcaaa agctcaagtt
tcgtgagccc cagcgtgagg 3300agaggatttg tcttgtcacg actaacttcc agaccaaatc
tatgtctagc atggtcagcg 3360atacctcgtg cacttttcca agcggcgatg gaatcttttg
gaagcactgg attcagacaa 3420aggacggcca atgcggttct cctctcgtaa gtacgcgcga
cggattcatc gtgggtattc 3480actccgcttc caacttcacc aacaccaaca actatttcac
tagcgtgcca aagaatttca 3540tggaattgct caccaaccag gaggcccaac aatgggttag
tggttggcgt cttaatgcgg 3600actcagtgct gtggggaggc cataaagttt tcatgaataa
gccggaggaa ccttttcaac 3660ccgtgaagga agcaacacag ctcatgaatg agctggttta
ctcacagtga taactcgagc 3720aatctgatac tagtaataaa agatgtttat tttcattaga
tgtgtgtgtt ggttttttgt 3780ctatagcatg c
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